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    Structured Review

    ATCC transfex
    Inverse expression between DEC1 and SHP (A) Suppressed expression of SHP by DEC1 HepG2 cells were seeded in 8-well chamber slides (2×10 4 cells/ well) and cultured for 24 h. Cells were then transfected with Flag-DEC1 (500 ng) or the vector by <t>TransfeX</t> reagent. The transfected cells were cultured for another 24 h and subsequently fixed with 4% paraformaldehyde for 10 min at pH 7.4. Cells were permeabilized with 0.1% Triton X-100 for 10 min. Chamber slides were incubated with 1% BSA (2mg/mL) for 1 h then incubated overnight with anti-SHP antibody or anti-Flag antibody. The anti-SHP antibody was located by Alexa Fluor ® 488 conjugated goat anti-rabbit IgG (green), whereas the anti-Flag antibody with Alexa Fluor ® 555 conjugated goat anti-mouse IgG (red). The slides were then mounted with ProLong Gold Antifade Mountant with DAPI (Blue). Cells were then imaged with confocal microscope. To provide more quantitative information, same experiments were performed in 6-well plates and cell lysates were prepared. Lysates (10 µg) were analyzed by Western blotting for the expression of SHP and DEC1 (Right panel). (B) Regulated expression of DEC1 and SHP by valproate in serum-shocked circadian induction HepG2 cells were seeded in 6- or 24-well plates at a density of 6×10 5 or 1.5×10 5 cells/well and cultured to reach 75% confluency. Cells were then shocked with media containing 50% horse serum for 2 h. Thereafter, the shocked cells were cultured in normal media or the same media containing valproate at 2 mM. Cells were harvested at a 6 h interval. The expression of DEC1 and SHP was determined by RT-qPCR with Taqman probes.
    Transfex, supplied by ATCC, used in various techniques. Bioz Stars score: 95/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "CIRCADIAN RHYTHMICITY: A FUNCTIONAL CONNECTION BETWEEN DIFFERENTIATED EMBRYONIC CHONDROCYTE-1 (DEC1) AND SMALL HETERODIMER PARTNER (SHP)"

    Article Title: CIRCADIAN RHYTHMICITY: A FUNCTIONAL CONNECTION BETWEEN DIFFERENTIATED EMBRYONIC CHONDROCYTE-1 (DEC1) AND SMALL HETERODIMER PARTNER (SHP)

    Journal: Archives of biochemistry and biophysics

    doi: 10.1016/j.abb.2017.08.004

    Inverse expression between DEC1 and SHP (A) Suppressed expression of SHP by DEC1 HepG2 cells were seeded in 8-well chamber slides (2×10 4 cells/ well) and cultured for 24 h. Cells were then transfected with Flag-DEC1 (500 ng) or the vector by TransfeX reagent. The transfected cells were cultured for another 24 h and subsequently fixed with 4% paraformaldehyde for 10 min at pH 7.4. Cells were permeabilized with 0.1% Triton X-100 for 10 min. Chamber slides were incubated with 1% BSA (2mg/mL) for 1 h then incubated overnight with anti-SHP antibody or anti-Flag antibody. The anti-SHP antibody was located by Alexa Fluor ® 488 conjugated goat anti-rabbit IgG (green), whereas the anti-Flag antibody with Alexa Fluor ® 555 conjugated goat anti-mouse IgG (red). The slides were then mounted with ProLong Gold Antifade Mountant with DAPI (Blue). Cells were then imaged with confocal microscope. To provide more quantitative information, same experiments were performed in 6-well plates and cell lysates were prepared. Lysates (10 µg) were analyzed by Western blotting for the expression of SHP and DEC1 (Right panel). (B) Regulated expression of DEC1 and SHP by valproate in serum-shocked circadian induction HepG2 cells were seeded in 6- or 24-well plates at a density of 6×10 5 or 1.5×10 5 cells/well and cultured to reach 75% confluency. Cells were then shocked with media containing 50% horse serum for 2 h. Thereafter, the shocked cells were cultured in normal media or the same media containing valproate at 2 mM. Cells were harvested at a 6 h interval. The expression of DEC1 and SHP was determined by RT-qPCR with Taqman probes.
    Figure Legend Snippet: Inverse expression between DEC1 and SHP (A) Suppressed expression of SHP by DEC1 HepG2 cells were seeded in 8-well chamber slides (2×10 4 cells/ well) and cultured for 24 h. Cells were then transfected with Flag-DEC1 (500 ng) or the vector by TransfeX reagent. The transfected cells were cultured for another 24 h and subsequently fixed with 4% paraformaldehyde for 10 min at pH 7.4. Cells were permeabilized with 0.1% Triton X-100 for 10 min. Chamber slides were incubated with 1% BSA (2mg/mL) for 1 h then incubated overnight with anti-SHP antibody or anti-Flag antibody. The anti-SHP antibody was located by Alexa Fluor ® 488 conjugated goat anti-rabbit IgG (green), whereas the anti-Flag antibody with Alexa Fluor ® 555 conjugated goat anti-mouse IgG (red). The slides were then mounted with ProLong Gold Antifade Mountant with DAPI (Blue). Cells were then imaged with confocal microscope. To provide more quantitative information, same experiments were performed in 6-well plates and cell lysates were prepared. Lysates (10 µg) were analyzed by Western blotting for the expression of SHP and DEC1 (Right panel). (B) Regulated expression of DEC1 and SHP by valproate in serum-shocked circadian induction HepG2 cells were seeded in 6- or 24-well plates at a density of 6×10 5 or 1.5×10 5 cells/well and cultured to reach 75% confluency. Cells were then shocked with media containing 50% horse serum for 2 h. Thereafter, the shocked cells were cultured in normal media or the same media containing valproate at 2 mM. Cells were harvested at a 6 h interval. The expression of DEC1 and SHP was determined by RT-qPCR with Taqman probes.

    Techniques Used: Expressing, Cell Culture, Transfection, Plasmid Preparation, Incubation, Microscopy, Western Blot, Quantitative RT-PCR

    2) Product Images from "CRISPR/Cas9 engineering of a KIM-1 reporter human proximal tubule cell line"

    Article Title: CRISPR/Cas9 engineering of a KIM-1 reporter human proximal tubule cell line

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0204487

    Lack of Cas9-mediated gene tagging of KIM-1 in RPTEC cells. RPTEC cells were transfected with jetPRIME ( A ), FuGENE-6 ( B ), and TransfeX ( C ) with TransfeX proving to be the most efficient. Despite good transfection efficiency, only 4 puromycin resistant clones were isolated. None of these clones demonstrated verified gene tagging via PCR ( D ). HK-2 clone 5 (HK-2-5) was used as a positive control. (-), untransfected cells.
    Figure Legend Snippet: Lack of Cas9-mediated gene tagging of KIM-1 in RPTEC cells. RPTEC cells were transfected with jetPRIME ( A ), FuGENE-6 ( B ), and TransfeX ( C ) with TransfeX proving to be the most efficient. Despite good transfection efficiency, only 4 puromycin resistant clones were isolated. None of these clones demonstrated verified gene tagging via PCR ( D ). HK-2 clone 5 (HK-2-5) was used as a positive control. (-), untransfected cells.

    Techniques Used: Transfection, Clone Assay, Isolation, Polymerase Chain Reaction, Positive Control

    3) Product Images from "CRISPR/Cas9 engineering of a KIM-1 reporter human proximal tubule cell line"

    Article Title: CRISPR/Cas9 engineering of a KIM-1 reporter human proximal tubule cell line

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0204487

    Lack of Cas9-mediated gene tagging of KIM-1 in RPTEC cells. RPTEC cells were transfected with jetPRIME ( A ), FuGENE-6 ( B ), and TransfeX ( C ) with TransfeX proving to be the most efficient. Despite good transfection efficiency, only 4 puromycin resistant clones were isolated. None of these clones demonstrated verified gene tagging via PCR ( D ). HK-2 clone 5 (HK-2-5) was used as a positive control. (-), untransfected cells.
    Figure Legend Snippet: Lack of Cas9-mediated gene tagging of KIM-1 in RPTEC cells. RPTEC cells were transfected with jetPRIME ( A ), FuGENE-6 ( B ), and TransfeX ( C ) with TransfeX proving to be the most efficient. Despite good transfection efficiency, only 4 puromycin resistant clones were isolated. None of these clones demonstrated verified gene tagging via PCR ( D ). HK-2 clone 5 (HK-2-5) was used as a positive control. (-), untransfected cells.

    Techniques Used: Transfection, Clone Assay, Isolation, Polymerase Chain Reaction, Positive Control

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    ATCC transfex
    Inverse expression between DEC1 and SHP (A) Suppressed expression of SHP by DEC1 HepG2 cells were seeded in 8-well chamber slides (2×10 4 cells/ well) and cultured for 24 h. Cells were then transfected with Flag-DEC1 (500 ng) or the vector by <t>TransfeX</t> reagent. The transfected cells were cultured for another 24 h and subsequently fixed with 4% paraformaldehyde for 10 min at pH 7.4. Cells were permeabilized with 0.1% Triton X-100 for 10 min. Chamber slides were incubated with 1% BSA (2mg/mL) for 1 h then incubated overnight with anti-SHP antibody or anti-Flag antibody. The anti-SHP antibody was located by Alexa Fluor ® 488 conjugated goat anti-rabbit IgG (green), whereas the anti-Flag antibody with Alexa Fluor ® 555 conjugated goat anti-mouse IgG (red). The slides were then mounted with ProLong Gold Antifade Mountant with DAPI (Blue). Cells were then imaged with confocal microscope. To provide more quantitative information, same experiments were performed in 6-well plates and cell lysates were prepared. Lysates (10 µg) were analyzed by Western blotting for the expression of SHP and DEC1 (Right panel). (B) Regulated expression of DEC1 and SHP by valproate in serum-shocked circadian induction HepG2 cells were seeded in 6- or 24-well plates at a density of 6×10 5 or 1.5×10 5 cells/well and cultured to reach 75% confluency. Cells were then shocked with media containing 50% horse serum for 2 h. Thereafter, the shocked cells were cultured in normal media or the same media containing valproate at 2 mM. Cells were harvested at a 6 h interval. The expression of DEC1 and SHP was determined by RT-qPCR with Taqman probes.
    Transfex, supplied by ATCC, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC lactobacillus buchneri
    The effect of exogenous lactobacilli addition on the viable number of lactobacilli in equine fecal cell suspensions. Grain types included minimally processed, finely ground (2 mm screen) corn, oats and wheat at 1.6% w/v starch concentration. Treatments included initial (open bars; 0 h), control (black bars; substrate only), and the addition of L . acidophilus (green bars), L . <t>buchneri</t> (blue bars), L . reuteri (red bars) and Mixed (purple bars; all 3 at equal concentrations) at 10 7 final concentration live or dead (autoclaved; corn only). Samples were taken after 24 h of incubation (37°C) for bacterial enumeration. The enumerations were performed on Rogosa SL agar (BD). The plates were incubated aerobically (37°C, 3 d). Plates with 30
    Lactobacillus Buchneri, supplied by ATCC, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Inverse expression between DEC1 and SHP (A) Suppressed expression of SHP by DEC1 HepG2 cells were seeded in 8-well chamber slides (2×10 4 cells/ well) and cultured for 24 h. Cells were then transfected with Flag-DEC1 (500 ng) or the vector by TransfeX reagent. The transfected cells were cultured for another 24 h and subsequently fixed with 4% paraformaldehyde for 10 min at pH 7.4. Cells were permeabilized with 0.1% Triton X-100 for 10 min. Chamber slides were incubated with 1% BSA (2mg/mL) for 1 h then incubated overnight with anti-SHP antibody or anti-Flag antibody. The anti-SHP antibody was located by Alexa Fluor ® 488 conjugated goat anti-rabbit IgG (green), whereas the anti-Flag antibody with Alexa Fluor ® 555 conjugated goat anti-mouse IgG (red). The slides were then mounted with ProLong Gold Antifade Mountant with DAPI (Blue). Cells were then imaged with confocal microscope. To provide more quantitative information, same experiments were performed in 6-well plates and cell lysates were prepared. Lysates (10 µg) were analyzed by Western blotting for the expression of SHP and DEC1 (Right panel). (B) Regulated expression of DEC1 and SHP by valproate in serum-shocked circadian induction HepG2 cells were seeded in 6- or 24-well plates at a density of 6×10 5 or 1.5×10 5 cells/well and cultured to reach 75% confluency. Cells were then shocked with media containing 50% horse serum for 2 h. Thereafter, the shocked cells were cultured in normal media or the same media containing valproate at 2 mM. Cells were harvested at a 6 h interval. The expression of DEC1 and SHP was determined by RT-qPCR with Taqman probes.

    Journal: Archives of biochemistry and biophysics

    Article Title: CIRCADIAN RHYTHMICITY: A FUNCTIONAL CONNECTION BETWEEN DIFFERENTIATED EMBRYONIC CHONDROCYTE-1 (DEC1) AND SMALL HETERODIMER PARTNER (SHP)

    doi: 10.1016/j.abb.2017.08.004

    Figure Lengend Snippet: Inverse expression between DEC1 and SHP (A) Suppressed expression of SHP by DEC1 HepG2 cells were seeded in 8-well chamber slides (2×10 4 cells/ well) and cultured for 24 h. Cells were then transfected with Flag-DEC1 (500 ng) or the vector by TransfeX reagent. The transfected cells were cultured for another 24 h and subsequently fixed with 4% paraformaldehyde for 10 min at pH 7.4. Cells were permeabilized with 0.1% Triton X-100 for 10 min. Chamber slides were incubated with 1% BSA (2mg/mL) for 1 h then incubated overnight with anti-SHP antibody or anti-Flag antibody. The anti-SHP antibody was located by Alexa Fluor ® 488 conjugated goat anti-rabbit IgG (green), whereas the anti-Flag antibody with Alexa Fluor ® 555 conjugated goat anti-mouse IgG (red). The slides were then mounted with ProLong Gold Antifade Mountant with DAPI (Blue). Cells were then imaged with confocal microscope. To provide more quantitative information, same experiments were performed in 6-well plates and cell lysates were prepared. Lysates (10 µg) were analyzed by Western blotting for the expression of SHP and DEC1 (Right panel). (B) Regulated expression of DEC1 and SHP by valproate in serum-shocked circadian induction HepG2 cells were seeded in 6- or 24-well plates at a density of 6×10 5 or 1.5×10 5 cells/well and cultured to reach 75% confluency. Cells were then shocked with media containing 50% horse serum for 2 h. Thereafter, the shocked cells were cultured in normal media or the same media containing valproate at 2 mM. Cells were harvested at a 6 h interval. The expression of DEC1 and SHP was determined by RT-qPCR with Taqman probes.

    Article Snippet: HepG2 Cells were transfected with DEC1 (Flag-tagged) by TransfeX (ATCC, Manassas, VA) for 24 h, washed and underwent cross-linking for 15 min by 1.0% formaldehyde at room temperature, and the cross-linking was terminated with glycine (final concentration of 125 mM).

    Techniques: Expressing, Cell Culture, Transfection, Plasmid Preparation, Incubation, Microscopy, Western Blot, Quantitative RT-PCR

    Overexpression of RNF168 and RNF8 reduces clonogenicity in WT but not in Foxm1 -deficient MEFs. WT and Foxm1 -deficient MEFs were transfected with the control pcDNA3, HA-RNF8 or Flag-RNF168. Twenty-four hours after transfection, the 2000 cells were seeded in six-well plates, treated with 0, 5 or 10 n m of epirubicn, grown for 15 days and then stained with crystal violet (top panel). The result (bottom panel) represents average of three independent experiments±s.d. Statistical significance was determined by Student's t -test (* P ⩽0.05, *** P ⩽0.005; n.s., non-significant).

    Journal: Oncogenesis

    Article Title: RNF168 cooperates with RNF8 to mediate FOXM1 ubiquitination and degradation in breast cancer epirubicin treatment

    doi: 10.1038/oncsis.2016.57

    Figure Lengend Snippet: Overexpression of RNF168 and RNF8 reduces clonogenicity in WT but not in Foxm1 -deficient MEFs. WT and Foxm1 -deficient MEFs were transfected with the control pcDNA3, HA-RNF8 or Flag-RNF168. Twenty-four hours after transfection, the 2000 cells were seeded in six-well plates, treated with 0, 5 or 10 n m of epirubicn, grown for 15 days and then stained with crystal violet (top panel). The result (bottom panel) represents average of three independent experiments±s.d. Statistical significance was determined by Student's t -test (* P ⩽0.05, *** P ⩽0.005; n.s., non-significant).

    Article Snippet: Cell culture, plasmids and transfection reagents The MCF-7 cell line used originated from the American Type Culture Collection and was acquired through CRUK cell bank (London, UK).

    Techniques: Over Expression, Transfection, Staining

    Overexpression of RNF168, but not RNF4 and RNF8, effectively repressed FOXM1 transcriptional activity. ( a ) Schematic showing of the WT (pGL2- Cyclin B1 -WT) and the FOXM1-mutant (pGL2- Cyclin B1 -mut) Cyclin B1 promoter/reporter. ( b ) MCF-7 cells were co-transfected with the WT cyclin B1 promoter (pGL2- Cyclin B1 -WT) and 20 ng of the empty expression vector (pcDNA3), Flag-RNF4, HA-RNF8 or Flag-RNF168 expression vector. Forty-eight hours after transfection, the transfected cells were western blotted for the expression of FOXM1, RNF168, RBF8, RNF4 and β-tubulin. ( c ) MCF-7 cells were co-transfected with the luciferase reporter driven by the WT cyclin B1 promoter (pGL2- Cyclin B1 -WT) and the empty expression vector (pcDNA3) or the Flag-RNF4, HA-RNF8 or Flag-RNF168 expression vector (0–50 ng) for 48 h as in ( b ). Reporter gene activity was expressed as a ratio of firefly luciferase activity to control Renilla luciferase activity. The promoter activity assay results are presented as bars representing mean±s.d. of three independent experiments in triplicates. All DNA concentrations were normalized using empty vector. ( d ) MCF-7 cells were co-transfected with Flag-RNF168 expression vector (0, 5, 10, 20, 30 and 50 ng) and the luciferase reporter driven by the WT cyclin B1 promoter (pGL2- Cyclin B1 -WT) or the mutant cyclin B1 promoter (pGL2- Cyclin B1 -mut) and the Flag-RNF168 expression vector (0–50 ng) for 48 h. Reporter gene activity was expressed as a ratio of firefly luciferase activity to control Renilla luciferase activity. The promoter activity assay results are presented as bars representing mean±s.d. of three independent experiments in triplicates. ** P ⩽0.01, *** P ⩽0.001 and ns indicates no significance by Student's t -test.

    Journal: Oncogenesis

    Article Title: RNF168 cooperates with RNF8 to mediate FOXM1 ubiquitination and degradation in breast cancer epirubicin treatment

    doi: 10.1038/oncsis.2016.57

    Figure Lengend Snippet: Overexpression of RNF168, but not RNF4 and RNF8, effectively repressed FOXM1 transcriptional activity. ( a ) Schematic showing of the WT (pGL2- Cyclin B1 -WT) and the FOXM1-mutant (pGL2- Cyclin B1 -mut) Cyclin B1 promoter/reporter. ( b ) MCF-7 cells were co-transfected with the WT cyclin B1 promoter (pGL2- Cyclin B1 -WT) and 20 ng of the empty expression vector (pcDNA3), Flag-RNF4, HA-RNF8 or Flag-RNF168 expression vector. Forty-eight hours after transfection, the transfected cells were western blotted for the expression of FOXM1, RNF168, RBF8, RNF4 and β-tubulin. ( c ) MCF-7 cells were co-transfected with the luciferase reporter driven by the WT cyclin B1 promoter (pGL2- Cyclin B1 -WT) and the empty expression vector (pcDNA3) or the Flag-RNF4, HA-RNF8 or Flag-RNF168 expression vector (0–50 ng) for 48 h as in ( b ). Reporter gene activity was expressed as a ratio of firefly luciferase activity to control Renilla luciferase activity. The promoter activity assay results are presented as bars representing mean±s.d. of three independent experiments in triplicates. All DNA concentrations were normalized using empty vector. ( d ) MCF-7 cells were co-transfected with Flag-RNF168 expression vector (0, 5, 10, 20, 30 and 50 ng) and the luciferase reporter driven by the WT cyclin B1 promoter (pGL2- Cyclin B1 -WT) or the mutant cyclin B1 promoter (pGL2- Cyclin B1 -mut) and the Flag-RNF168 expression vector (0–50 ng) for 48 h. Reporter gene activity was expressed as a ratio of firefly luciferase activity to control Renilla luciferase activity. The promoter activity assay results are presented as bars representing mean±s.d. of three independent experiments in triplicates. ** P ⩽0.01, *** P ⩽0.001 and ns indicates no significance by Student's t -test.

    Article Snippet: Cell culture, plasmids and transfection reagents The MCF-7 cell line used originated from the American Type Culture Collection and was acquired through CRUK cell bank (London, UK).

    Techniques: Over Expression, Activity Assay, Mutagenesis, Transfection, Expressing, Plasmid Preparation, Western Blot, Luciferase

    FOXM1 is modified by ubiquitination and its K48-linked polyubiquitin conjugates can be induced by RNF168 and epirubicin. ( a ) 293T cells were co-transfected with eGFP-FOXM1 and empty control expression vector, His-Ubiquitin(K48R) or His-Ubiquitin(K63R). Ubiquitinated proteins were purified using Ni 2+ -column affinity pull-down under denaturing conditions (Guanidinium Chloride). Input and His-tagged Ubiquitin pulled-down proteins were probed for FOXM1 using an anti-FOXM1 antibody. The result showed that higher levels of His-Ub(K63R) were conjugated to FOXM1, suggesting that FOXM1 is primarily covalently linked to K48 polyubiquitin conjugates. ( b ) 293T cells were co-transfected with eGFP-FOXM1 and empty control expression vector or His-Ubiquitin and in the absence or presence of Flag-RNF168. Ubiquitinated proteins were purified using Ni 2+ -column affinity pull-down under denaturing conditions. The input (1/10) and pulled-down proteins were western blotted for FOXM1 expression. The results showed that RNF168 overexpression can further enhance FOXM1 ubiquitination. ( c ) 293T cells were co-transfected with His-Ub(K63R) in the presence or absence of HA-RNF8 or Flag-168. Ubiquitinated proteins were purified under denaturing conditions and probed with FOXM1. The results suggested that RNF168, but not RNF8, can induce FOXM1-K48 polyubiquitination. ( d ) 293T cells were co-transfected with His-Ub and eGFP-FOXM-1 (WT) or eGFP-FOXM1-5X(K > R). His-tagged ubiquitinated proteins were purified under denaturing conditions. The pulled-down His-Ub conjugated proteins were western blotted for FOXM1 expression. The results showed that substantially higher levels of eGFP-FOXM1 WT proteins were pulled down with the His-Ub than the SUMOylation-defective mutant, despite the mutant being the more stable species, suggesting that SUMOylation promotes FOXM1 ubiquitination. ( e ) MCF-7 cells were transfected with His-FOXM1 and Flag-RNF168 or control empty expression vector. Twenty-four hours after transfection, the cells were lysed, the His-tagged FOXM1 purified using Ni 2+ -column affinity pull-down under denaturing conditions and probed with FOXM1, specific K48-linked and K63-linked polyubiquitin antibodies. The results indicated that RNF168 predominantly enhances FOXM1-K48 polyubiquitination in untreated and epirubicin-treated MCF-7 cells.

    Journal: Oncogenesis

    Article Title: RNF168 cooperates with RNF8 to mediate FOXM1 ubiquitination and degradation in breast cancer epirubicin treatment

    doi: 10.1038/oncsis.2016.57

    Figure Lengend Snippet: FOXM1 is modified by ubiquitination and its K48-linked polyubiquitin conjugates can be induced by RNF168 and epirubicin. ( a ) 293T cells were co-transfected with eGFP-FOXM1 and empty control expression vector, His-Ubiquitin(K48R) or His-Ubiquitin(K63R). Ubiquitinated proteins were purified using Ni 2+ -column affinity pull-down under denaturing conditions (Guanidinium Chloride). Input and His-tagged Ubiquitin pulled-down proteins were probed for FOXM1 using an anti-FOXM1 antibody. The result showed that higher levels of His-Ub(K63R) were conjugated to FOXM1, suggesting that FOXM1 is primarily covalently linked to K48 polyubiquitin conjugates. ( b ) 293T cells were co-transfected with eGFP-FOXM1 and empty control expression vector or His-Ubiquitin and in the absence or presence of Flag-RNF168. Ubiquitinated proteins were purified using Ni 2+ -column affinity pull-down under denaturing conditions. The input (1/10) and pulled-down proteins were western blotted for FOXM1 expression. The results showed that RNF168 overexpression can further enhance FOXM1 ubiquitination. ( c ) 293T cells were co-transfected with His-Ub(K63R) in the presence or absence of HA-RNF8 or Flag-168. Ubiquitinated proteins were purified under denaturing conditions and probed with FOXM1. The results suggested that RNF168, but not RNF8, can induce FOXM1-K48 polyubiquitination. ( d ) 293T cells were co-transfected with His-Ub and eGFP-FOXM-1 (WT) or eGFP-FOXM1-5X(K > R). His-tagged ubiquitinated proteins were purified under denaturing conditions. The pulled-down His-Ub conjugated proteins were western blotted for FOXM1 expression. The results showed that substantially higher levels of eGFP-FOXM1 WT proteins were pulled down with the His-Ub than the SUMOylation-defective mutant, despite the mutant being the more stable species, suggesting that SUMOylation promotes FOXM1 ubiquitination. ( e ) MCF-7 cells were transfected with His-FOXM1 and Flag-RNF168 or control empty expression vector. Twenty-four hours after transfection, the cells were lysed, the His-tagged FOXM1 purified using Ni 2+ -column affinity pull-down under denaturing conditions and probed with FOXM1, specific K48-linked and K63-linked polyubiquitin antibodies. The results indicated that RNF168 predominantly enhances FOXM1-K48 polyubiquitination in untreated and epirubicin-treated MCF-7 cells.

    Article Snippet: Cell culture, plasmids and transfection reagents The MCF-7 cell line used originated from the American Type Culture Collection and was acquired through CRUK cell bank (London, UK).

    Techniques: Modification, Transfection, Expressing, Plasmid Preparation, Purification, Affinity Column, Western Blot, Over Expression, Mutagenesis

    RNF168 negatively regulates FOXM1 expression and cell proliferation in MCF-7 cells. ( a ) Western blot analysis was performed on MCF-7 cells transfected with empty expression vector (pcDNA3) and Flag-RNF4, HA-RNF8 or Flag-RNF168 expression vector for 48 h. The protein expression levels of FOXM1, the RNF E3-ligase and β-tubulin were assessed by western blot analysis (asterisk indicates the non-specific protein band). FOXM1 expression was observed to be downregulated by RNF168, but not by RNF4 and RNF8, at the protein level. ( b ) MCF-7 cells were transfected with NSC siRNA, or with RNF4, RNF8 or RNF168 specific siRNA pool for 48 h. The protein expression levels of FOXM1, the RNF E3-ligase and β-tubulin were assessed by western blot analysis. FOXM1 expression was observed to be upregulated by the silencing of RNF168, but not RNF4 and RNF8, at the protein level. ( c ) MCF-7 cells were either untransfected (Mock), or transfected with pcDNA3-empty vector, with RNF168 or with Flag-RNF168 for 48 h. Western blot analysis was performed on the transfected MCF-7 cells with or without 1 μ m epirubicin treatment for 24 h. The protein expression levels of FOXM1, RNF168, Flag-(RNF168) and β-tubulin were assessed by western blot analysis. ( d ) MCF-7 cells were transfected with NSC siRNA, siRNA pool targeting RNF168, pcDNA3-empty vector or pcDNA3-Flag-RNF168. Twenty-four hours after transfection, aliquots of the transfected cells were split into 96-well plates with concentrations of epirubicin indicated and their proliferation analysed at 48 h by SRB assays. Cell proliferation assays revealed that Flag-RNF168 overexpression increased and RNF168 depletion with siRNA decreased epirubicin sensitivity in MCF-7 cells. The results presented as bars representing mean±s.d. of three independent experiments in triplicates. The differences between that RNF168 overexpression or depletion and their relative controls are significant at * P

    Journal: Oncogenesis

    Article Title: RNF168 cooperates with RNF8 to mediate FOXM1 ubiquitination and degradation in breast cancer epirubicin treatment

    doi: 10.1038/oncsis.2016.57

    Figure Lengend Snippet: RNF168 negatively regulates FOXM1 expression and cell proliferation in MCF-7 cells. ( a ) Western blot analysis was performed on MCF-7 cells transfected with empty expression vector (pcDNA3) and Flag-RNF4, HA-RNF8 or Flag-RNF168 expression vector for 48 h. The protein expression levels of FOXM1, the RNF E3-ligase and β-tubulin were assessed by western blot analysis (asterisk indicates the non-specific protein band). FOXM1 expression was observed to be downregulated by RNF168, but not by RNF4 and RNF8, at the protein level. ( b ) MCF-7 cells were transfected with NSC siRNA, or with RNF4, RNF8 or RNF168 specific siRNA pool for 48 h. The protein expression levels of FOXM1, the RNF E3-ligase and β-tubulin were assessed by western blot analysis. FOXM1 expression was observed to be upregulated by the silencing of RNF168, but not RNF4 and RNF8, at the protein level. ( c ) MCF-7 cells were either untransfected (Mock), or transfected with pcDNA3-empty vector, with RNF168 or with Flag-RNF168 for 48 h. Western blot analysis was performed on the transfected MCF-7 cells with or without 1 μ m epirubicin treatment for 24 h. The protein expression levels of FOXM1, RNF168, Flag-(RNF168) and β-tubulin were assessed by western blot analysis. ( d ) MCF-7 cells were transfected with NSC siRNA, siRNA pool targeting RNF168, pcDNA3-empty vector or pcDNA3-Flag-RNF168. Twenty-four hours after transfection, aliquots of the transfected cells were split into 96-well plates with concentrations of epirubicin indicated and their proliferation analysed at 48 h by SRB assays. Cell proliferation assays revealed that Flag-RNF168 overexpression increased and RNF168 depletion with siRNA decreased epirubicin sensitivity in MCF-7 cells. The results presented as bars representing mean±s.d. of three independent experiments in triplicates. The differences between that RNF168 overexpression or depletion and their relative controls are significant at * P

    Article Snippet: Cell culture, plasmids and transfection reagents The MCF-7 cell line used originated from the American Type Culture Collection and was acquired through CRUK cell bank (London, UK).

    Techniques: Expressing, Western Blot, Transfection, Plasmid Preparation, Sulforhodamine B Assay, Over Expression

    Lack of Cas9-mediated gene tagging of KIM-1 in RPTEC cells. RPTEC cells were transfected with jetPRIME ( A ), FuGENE-6 ( B ), and TransfeX ( C ) with TransfeX proving to be the most efficient. Despite good transfection efficiency, only 4 puromycin resistant clones were isolated. None of these clones demonstrated verified gene tagging via PCR ( D ). HK-2 clone 5 (HK-2-5) was used as a positive control. (-), untransfected cells.

    Journal: PLoS ONE

    Article Title: CRISPR/Cas9 engineering of a KIM-1 reporter human proximal tubule cell line

    doi: 10.1371/journal.pone.0204487

    Figure Lengend Snippet: Lack of Cas9-mediated gene tagging of KIM-1 in RPTEC cells. RPTEC cells were transfected with jetPRIME ( A ), FuGENE-6 ( B ), and TransfeX ( C ) with TransfeX proving to be the most efficient. Despite good transfection efficiency, only 4 puromycin resistant clones were isolated. None of these clones demonstrated verified gene tagging via PCR ( D ). HK-2 clone 5 (HK-2-5) was used as a positive control. (-), untransfected cells.

    Article Snippet: RPTEC/TERT1 cells were transfected with FuGENE 6, jetPRIME (VWR, Radnor PA), or TransfeX (ATCC, Manassas, VA) the following day according to the manufacturer’s instructions.

    Techniques: Transfection, Clone Assay, Isolation, Polymerase Chain Reaction, Positive Control

    The effect of exogenous lactobacilli addition on the viable number of lactobacilli in equine fecal cell suspensions. Grain types included minimally processed, finely ground (2 mm screen) corn, oats and wheat at 1.6% w/v starch concentration. Treatments included initial (open bars; 0 h), control (black bars; substrate only), and the addition of L . acidophilus (green bars), L . buchneri (blue bars), L . reuteri (red bars) and Mixed (purple bars; all 3 at equal concentrations) at 10 7 final concentration live or dead (autoclaved; corn only). Samples were taken after 24 h of incubation (37°C) for bacterial enumeration. The enumerations were performed on Rogosa SL agar (BD). The plates were incubated aerobically (37°C, 3 d). Plates with 30

    Journal: PLoS ONE

    Article Title: Exogenous lactobacilli mitigate microbial changes associated with grain fermentation (corn, oats, and wheat) by equine fecal microflora ex vivo

    doi: 10.1371/journal.pone.0174059

    Figure Lengend Snippet: The effect of exogenous lactobacilli addition on the viable number of lactobacilli in equine fecal cell suspensions. Grain types included minimally processed, finely ground (2 mm screen) corn, oats and wheat at 1.6% w/v starch concentration. Treatments included initial (open bars; 0 h), control (black bars; substrate only), and the addition of L . acidophilus (green bars), L . buchneri (blue bars), L . reuteri (red bars) and Mixed (purple bars; all 3 at equal concentrations) at 10 7 final concentration live or dead (autoclaved; corn only). Samples were taken after 24 h of incubation (37°C) for bacterial enumeration. The enumerations were performed on Rogosa SL agar (BD). The plates were incubated aerobically (37°C, 3 d). Plates with 30

    Article Snippet: Effect of exogenous lactobacilli addition and concentration on pH Lactobacillus acidophilus (ATCC # 4356), Lactobacillus buchneri (ATCC # 4005) and Lactobacillus reuteri (ATCC # 23272) type strains were obtained from the American Type Culture Collection (Manassas, VA, USA).

    Techniques: Concentration Assay, Incubation

    The effect of exogenous lactobacilli addition on the viable number of total amylolytic bacteria in equine fecal cell suspensions. Grain types included minimally processed, finely ground (2 mm screen) corn, oats and wheat at 1.6% w/v starch concentration. Treatments included initial (open bars; 0 h), control (black bars; substrate only), and the addition of L . acidophilus (green bars), L . buchneri (blue bars), L . reuteri (red bars) and Mixed (purple bars; all 3 at equal concentrations) at 10 7 final concentration live or dead (autoclaved; corn only). Samples were taken after 24 h of incubation (37°C) for bacterial enumeration. The enumerations were performed in anaerobic liquid media with soluble starch as the growth substrate. The tubes were incubated (37°C, 3 d), and the final dilution exhibiting bacterial growth (visual examination) was recorded as the viable number. Hatched lines separate individual statistical comparisons. Means lacking a common letter are different between treatments within substrate ( P

    Journal: PLoS ONE

    Article Title: Exogenous lactobacilli mitigate microbial changes associated with grain fermentation (corn, oats, and wheat) by equine fecal microflora ex vivo

    doi: 10.1371/journal.pone.0174059

    Figure Lengend Snippet: The effect of exogenous lactobacilli addition on the viable number of total amylolytic bacteria in equine fecal cell suspensions. Grain types included minimally processed, finely ground (2 mm screen) corn, oats and wheat at 1.6% w/v starch concentration. Treatments included initial (open bars; 0 h), control (black bars; substrate only), and the addition of L . acidophilus (green bars), L . buchneri (blue bars), L . reuteri (red bars) and Mixed (purple bars; all 3 at equal concentrations) at 10 7 final concentration live or dead (autoclaved; corn only). Samples were taken after 24 h of incubation (37°C) for bacterial enumeration. The enumerations were performed in anaerobic liquid media with soluble starch as the growth substrate. The tubes were incubated (37°C, 3 d), and the final dilution exhibiting bacterial growth (visual examination) was recorded as the viable number. Hatched lines separate individual statistical comparisons. Means lacking a common letter are different between treatments within substrate ( P

    Article Snippet: Effect of exogenous lactobacilli addition and concentration on pH Lactobacillus acidophilus (ATCC # 4356), Lactobacillus buchneri (ATCC # 4005) and Lactobacillus reuteri (ATCC # 23272) type strains were obtained from the American Type Culture Collection (Manassas, VA, USA).

    Techniques: Concentration Assay, Incubation

    The effect of exogenous lactobacilli addition on the pH of equine fecal cell suspensions. Grain types included minimally processed, finely ground (2 mm screen) corn, oats and wheat at 1.6% w/v starch concentration. Treatments included initial (open bars; 0 h), control (black bars; substrate only), and the addition of L . acidophilus (green bars), L . buchneri (blue bars), L . reuteri (red bars) and Mixed (purple bars; all 3 at equal concentrations) at 10 7 final concentration live or dead (autoclaved; corn only). Samples were taken after 24 h of incubation (37°C) for pH. Hatched lines separate individual statistical comparisons. Means lacking a common letter are different between treatments within substrate ( P

    Journal: PLoS ONE

    Article Title: Exogenous lactobacilli mitigate microbial changes associated with grain fermentation (corn, oats, and wheat) by equine fecal microflora ex vivo

    doi: 10.1371/journal.pone.0174059

    Figure Lengend Snippet: The effect of exogenous lactobacilli addition on the pH of equine fecal cell suspensions. Grain types included minimally processed, finely ground (2 mm screen) corn, oats and wheat at 1.6% w/v starch concentration. Treatments included initial (open bars; 0 h), control (black bars; substrate only), and the addition of L . acidophilus (green bars), L . buchneri (blue bars), L . reuteri (red bars) and Mixed (purple bars; all 3 at equal concentrations) at 10 7 final concentration live or dead (autoclaved; corn only). Samples were taken after 24 h of incubation (37°C) for pH. Hatched lines separate individual statistical comparisons. Means lacking a common letter are different between treatments within substrate ( P

    Article Snippet: Effect of exogenous lactobacilli addition and concentration on pH Lactobacillus acidophilus (ATCC # 4356), Lactobacillus buchneri (ATCC # 4005) and Lactobacillus reuteri (ATCC # 23272) type strains were obtained from the American Type Culture Collection (Manassas, VA, USA).

    Techniques: Concentration Assay, Incubation

    The effect of exogenous lactobacilli addition on the viable number of Group D Gram-positive cocci (GPC) in equine fecal cell suspensions. Grain types included minimally processed, finely ground (2 mm screen) corn, oats and wheat at 1.6% w/v starch concentration. Treatments included initial (open bars; 0 h), control (black bars; substrate only), and the addition of L . acidophilus (green bars), L . buchneri (blue bars), L . reuteri (red bars) and Mixed (purple bars; all 3 at equal concentrations) at 10 7 final concentration live or dead (autoclaved; corn only). Samples were taken after 24 h of incubation (37°C) for bacterial enumeration. GPC were enumerated on bile esculin azide agar (BD). The plates were incubated aerobically (37°C, 3 d). Plates with 30

    Journal: PLoS ONE

    Article Title: Exogenous lactobacilli mitigate microbial changes associated with grain fermentation (corn, oats, and wheat) by equine fecal microflora ex vivo

    doi: 10.1371/journal.pone.0174059

    Figure Lengend Snippet: The effect of exogenous lactobacilli addition on the viable number of Group D Gram-positive cocci (GPC) in equine fecal cell suspensions. Grain types included minimally processed, finely ground (2 mm screen) corn, oats and wheat at 1.6% w/v starch concentration. Treatments included initial (open bars; 0 h), control (black bars; substrate only), and the addition of L . acidophilus (green bars), L . buchneri (blue bars), L . reuteri (red bars) and Mixed (purple bars; all 3 at equal concentrations) at 10 7 final concentration live or dead (autoclaved; corn only). Samples were taken after 24 h of incubation (37°C) for bacterial enumeration. GPC were enumerated on bile esculin azide agar (BD). The plates were incubated aerobically (37°C, 3 d). Plates with 30

    Article Snippet: Effect of exogenous lactobacilli addition and concentration on pH Lactobacillus acidophilus (ATCC # 4356), Lactobacillus buchneri (ATCC # 4005) and Lactobacillus reuteri (ATCC # 23272) type strains were obtained from the American Type Culture Collection (Manassas, VA, USA).

    Techniques: Gel Permeation Chromatography, Concentration Assay, Incubation

    The effect of exogenous lactobacilli addition on the viable number of total lactate-utilizing bacteria in equine fecal cell suspensions. Grain types included minimally processed, finely ground (2 mm screen) corn, oats and wheat at 1.6% w/v starch concentration. Treatments included initial (open bars; 0 h), control (black bars; substrate only), and the addition of L . acidophilus (green bars), L . buchneri (blue bars), L . reuteri (red bars) and Mixed (purple bars; all 3 at equal concentrations) at 10 7 final concentration live or dead (autoclaved; corn only). Samples were taken after 24 h of incubation (37°C) for bacterial enumeration. Total lactate-utilizing bacteria were enumerated on L-U agar. The plates were incubated anaerobically (37°C, 5 d). Plates with 30

    Journal: PLoS ONE

    Article Title: Exogenous lactobacilli mitigate microbial changes associated with grain fermentation (corn, oats, and wheat) by equine fecal microflora ex vivo

    doi: 10.1371/journal.pone.0174059

    Figure Lengend Snippet: The effect of exogenous lactobacilli addition on the viable number of total lactate-utilizing bacteria in equine fecal cell suspensions. Grain types included minimally processed, finely ground (2 mm screen) corn, oats and wheat at 1.6% w/v starch concentration. Treatments included initial (open bars; 0 h), control (black bars; substrate only), and the addition of L . acidophilus (green bars), L . buchneri (blue bars), L . reuteri (red bars) and Mixed (purple bars; all 3 at equal concentrations) at 10 7 final concentration live or dead (autoclaved; corn only). Samples were taken after 24 h of incubation (37°C) for bacterial enumeration. Total lactate-utilizing bacteria were enumerated on L-U agar. The plates were incubated anaerobically (37°C, 5 d). Plates with 30

    Article Snippet: Effect of exogenous lactobacilli addition and concentration on pH Lactobacillus acidophilus (ATCC # 4356), Lactobacillus buchneri (ATCC # 4005) and Lactobacillus reuteri (ATCC # 23272) type strains were obtained from the American Type Culture Collection (Manassas, VA, USA).

    Techniques: Concentration Assay, Incubation