mission shrna library  (Merck KGaA)

Journal: Molecular Oncology

Article Title: Respiratory complex I‐mediated NAD + regeneration regulates cancer cell proliferation through the transcriptional and translational control of p21 Cip1 expression by SIRT3 and SIRT7

doi: 10.1002/1878-0261.13808

Figure Lengend Snippet: Complex I function mediated by NDUFV1 is important for cell cycle progression in cancer cells. (A) Western blotting with the indicated antibodies in MDA‐MB‐231 cells after 48 h of treatment with siRNAs for the major catalytic subunits of the electron transport chain complexes (I, NDUFV1 ; II, SDHA ; III, UQCRFS1 ; IV, SURF1 ). Ctr, negative control siRNA. α‐Tubulin is the loading control. Silencing efficiency of each siRNA was evaluated based on the band intensity quantified by imagej software, which was normalized to that of α‐tubulin and shown relative to Ctr. Representative results from three independent experiments are shown. (B, C) Cell proliferation and viability (B, MDA‐MB‐231 and MCF7; C, JHH‐4 and HLF) after transfection with the indicated siRNAs for 72 h: NDU, NDUFV1 . Silencing efficiencies of siRNAs are indicated in Table . (D, E) Doxycycline (DOX)‐responsive (TetOFF) shRNA [NDU (NDFUV1) or NT (nontarget control) (Table )]‐expressing cells were obtained from MCF7 cells by lentiviral transduction of the constructs (Materials and methods). Cells were incubated for 48 h in the presence (+) or absence (−) of DOX (1.0 μg·mL −1 ) and NDUFV1 knockdown was validated by western blotting. Representative blots from three independent experiments are shown (D). Growth curve and doubling time. 2 × 10 4 cells were seeded in the presence or absence of DOX; the number of viable cells was counted on each culture day. Doubling time (DT) was calculated from the slope of the graph (E). (F) Cell cycle distribution of siRNA‐transfected MCF7 and MDA‐MB‐231 cells. Bars and plots represent the means ± standard deviation from three independent experiments with measurements in triplicate in each experiment. For statistical analysis, two‐tailed Student's t ‐test (C and F) and one‐way ANOVA Dunnett's multiple comparisons test among group (B and E) were used; ** P < 0.01.

Article Snippet: Target sequences for nontargeting control and each gene were obtained from Mission shRNA library (Merck KGaA) (Table ). siRNA (FlexiTube siRNA) was purchased from Qiagen (Venlo, the Netherlands).

Techniques: Western Blot, Negative Control, Control, Software, Transfection, shRNA, Expressing, Transduction, Construct, Incubation, Knockdown, Standard Deviation, Two Tailed Test

Journal: Molecular Oncology

Article Title: Respiratory complex I‐mediated NAD + regeneration regulates cancer cell proliferation through the transcriptional and translational control of p21 Cip1 expression by SIRT3 and SIRT7

doi: 10.1002/1878-0261.13808

Figure Lengend Snippet: NDUFV1 downregulation upregulates p21 Cip1 expression in cancer cells. (A–C) Cells were incubated with siRNAs for 48 h: NDU, siRNA for NDUFV1 ; Ctr, negative control siRNA. Western blotting with the indicated antibodies. Band intensities measured with ImageJ are shown as relative to the control (Ctr). Representative blots from three independent experiments are shown. α‐Tubulin and β‐actin are the loading controls (A). The mRNA levels of p21 Cip1 examined using quantitative reverse transcription PCR (qRT‐PCR). Values are relative to control (Ctr) (B). Cells were treated at 0 h with actinomycin D (1 μg·mL −1 ) and at each time point, the amount of p21 Cip1 mRNA was determined by qRT‐PCR. The percentages of remaining p21 Cip1 mRNA were graphed (value at 0 h was defined as 100%). The half‐life ( t 1/2 ) of mRNA was calculated from the slope of the graph (C). (D) Reporter activity assessed in NDUFV1 ‐silenced MCF7 cells with shRNA or siRNA as in A and Fig. . Cells were transfected and incubated with WWP reporter plasmids (Materials and methods) together with the internal control for 48 h. A ratio to the control (NT, Ctr) is shown. Trichostatin A (TSA, 0.5 μ m ) was used as positive control. RLU, relative luciferase units. (E, F) DOX‐responsive (TetOFF) shRNA‐expressing MCF7 cells as in Fig. were transfected with siRNA for p21 Cip1 or negative control siRNA (Ctr) in the presence (+) or absence (−) of DOX (1 μg·mL −1 ). After 48 h, knockdown effects were validated as in A. Band intensities are shown as relative to control (shNT/Ctr, DOX+). Representative blots from three independent experiments are shown. α‐Tubulin is the loading control (E). Cell proliferation and viability were assessed after 72 h of transfection (F). Bars and plots represent the means ± standard deviation from three independent experiments with measurements in triplicate in each experiment. Two‐tailed Student's t ‐test (B and C) and one‐way ANOVA following by Dunnett's multiple comparisons test among group (D and F); ** P < 0.01, n.s., not significance.

Article Snippet: Target sequences for nontargeting control and each gene were obtained from Mission shRNA library (Merck KGaA) (Table ). siRNA (FlexiTube siRNA) was purchased from Qiagen (Venlo, the Netherlands).

Techniques: Expressing, Incubation, Negative Control, Western Blot, Control, Reverse Transcription, Quantitative RT-PCR, Activity Assay, shRNA, Transfection, Positive Control, Luciferase, Knockdown, Standard Deviation, Two Tailed Test

Journal: Molecular Oncology

Article Title: Respiratory complex I‐mediated NAD + regeneration regulates cancer cell proliferation through the transcriptional and translational control of p21 Cip1 expression by SIRT3 and SIRT7

doi: 10.1002/1878-0261.13808

Figure Lengend Snippet: Decrease in the NAD + /NADH ratio signals to upregulate p21 Cip1 expression when Complex I activity is silenced. (A) MCF7 cells constitutively expressing shNDUFV1 (NDU) #1 or #2 and nontarget control shRNA (NT) were established. Immediately after selection and confirmation of NDUFV1 knockdown by western blotting (GAPDH: the loading control), the cells were treated and extracted with detergents and subjected to Blue native PAGE followed by western blotting with anti‐NDUFV1 antibody (WB: NDUFV1) or in‐gel Complex I (CI) activity assay (Materials and methods). Representative results from two independent experiments are shown. (B–D) MCF7 cells were treated with siRNA (Ctr, control; NDU, NDUFV1 ) in the presence or absence of 1 m m β‐nicotinamide mononucleotide (NMN), and the NAD + /NADH ratio was evaluated after 24 h (B). The levels of protein (C) and mRNA (D) were examined after 48 h by western blotting and qRT‐PCR, respectively. Values are relative to control (Ctr/−). Representative blots from three independent experiments are shown. Arrow and arrowhead indicate the position of NDUFV1 and unknown contingent bands, respectively. α‐Tubulin is the loading control. (E–G) MCF7 cells were infected with DOX‐responsive lentivirus constructs (TetON) expressing Flag‐tagged native ( Lb NOX), mitochondrial‐targeted Lb NOX (mito Lb NOX), or control (Mock). After 48 h of transfection with siRNA with (+) or without (−) DOX (0.5 μg·mL −1 ), western blotting (E) and qRT‐PCR (G) were performed. Representative blots from three independent experiments are shown. β‐Actin is the loading control. Values are relative to control (Ctr/DOX−). The NAD + /NADH ratio was determined after treatment with siRNA for 24 h in the presence or absence of DOX (F). Bars represent the means ± standard deviation from three independent experiments with measurements in triplicate in each experiment. One‐way ANOVA with Bonferroni post hoc test among group; ** P < 0.01.

Article Snippet: Target sequences for nontargeting control and each gene were obtained from Mission shRNA library (Merck KGaA) (Table ). siRNA (FlexiTube siRNA) was purchased from Qiagen (Venlo, the Netherlands).

Techniques: Expressing, Activity Assay, Control, shRNA, Selection, Knockdown, Western Blot, Blue Native PAGE, Quantitative RT-PCR, Infection, Construct, Transfection, Standard Deviation

Journal: Molecular Oncology

Article Title: Respiratory complex I‐mediated NAD + regeneration regulates cancer cell proliferation through the transcriptional and translational control of p21 Cip1 expression by SIRT3 and SIRT7

doi: 10.1002/1878-0261.13808

Figure Lengend Snippet: Translation of p21 Cip1 is promoted by the downregulation of SIRT3 activity when Complex I activity is silenced. (A) Western blotting and qRT‐PCR with MCF7 cells transfected with siRNA for SIRT3 (silencing efficiency is indicated in Table ) or negative control siRNA (Ctr) for 48 h. Values are relative to control (Ctr). β‐Actin is the loading control. (B) Western blotting and qRT‐PCR with MCF7 cells expressing Flag‐tagged SIRT3 (Flag‐SIRT3) or control (Mock) after 48 h of transfection with NDUFV1 siRNA (NDU) or negative control siRNA (Ctr). Values are relative to control (Ctr). Arrowhead and arrow indicate exogenous and endogenous SIRT3, respectively. α‐Tubulin is the loading control. (C) MCF7 and MDA‐MB‐231 cells expressing DOX‐responsive (TetOFF) shRNA (Table ) were cultured for 72 h in the absence of DOX. After treatment with cycloheximide (100 μg·mL −1 ) for the indicated time, p21 Cip1 was quantified by western blotting. After normalization to α‐tubulin values, the remaining amounts were plotted (value at 0 h was defined as 100%). The half‐life ( t 1/2 ) of protein was calculated from the slope of the graph. α‐Tubulin is the loading control. (D) The acetylation levels of SOD2 (SOD2ac) were examined using western blotting in cells expressing Flag‐tagged SOD2 after 48 h of transfection with siRNA. The band intensity of SOD2ac was normalized to that of SOD2 and shown as relative to control (Ctr). α‐Tubulin and β‐actin are the loading controls. (E) MCF7 cells were transfected with the indicated siRNAs, and nascent protein synthesis was examined after 24 h (Materials and methods). The affinity‐purified fractions were examined using western blotting with the indicated antibodies. The band intensity of p21 Cip1 was normalized to that of β‐actin and shown as relative to control (Ctr). Representative results from three independent experiments are shown. β‐Actin is the loading control. In western blotting, representative blots from three independent experiments are shown. Bars and plots represent the means ± standard deviation from three independent experiments with measurements in triplicate in each experiment. Two‐tailed Student's t ‐test (A and C) and one‐way ANOVA following by Bonferroni multiple comparisons test among group (B); ** P < 0.01, n.s., not significance.

Article Snippet: Target sequences for nontargeting control and each gene were obtained from Mission shRNA library (Merck KGaA) (Table ). siRNA (FlexiTube siRNA) was purchased from Qiagen (Venlo, the Netherlands).

Techniques: Activity Assay, Western Blot, Quantitative RT-PCR, Transfection, Negative Control, Control, Expressing, shRNA, Cell Culture, Affinity Purification, Standard Deviation, Two Tailed Test

Journal: Molecular Oncology

Article Title: Respiratory complex I‐mediated NAD + regeneration regulates cancer cell proliferation through the transcriptional and translational control of p21 Cip1 expression by SIRT3 and SIRT7

doi: 10.1002/1878-0261.13808

Figure Lengend Snippet: Transcription of p21 Cip1 is suppressed by SIRT7 activity under Complex I activity. (A) Western blotting and qRT‐PCR performed in MCF7 cells after 48 h of transfection with siRNA for SIRT7 or negative control siRNA (Ctr). Values are relative to control (Ctr). Representative blots from three independent experiments are shown. β‐Actin is the loading control. (B–D) MCF7 cells expressing Flag‐tagged SIRT7 (wild‐type, SIRT7 WT; a catalytically inactive mutant, SIRT7 H187Y), or control (Mock) were treated with NDUFV1 (NDU) siRNA or negative control siRNA (Ctr). After 48 h, the mRNA levels of p21 Cip1 were examined by qRT‐PCR (B). Values are relative to control (Mock/Ctr). In (C), the WWP Pst reporter was transfected into cells with the internal control and siRNA, and after 48 h, reporter activities were evaluated as in Fig. . Fold changes normalized to the control (Ctr) are graphed. In (D), ChIP was performed to examine the enrichment of acetyl H3K18 on the p21 Cip1 locus after treatment with siRNA for 30 h. Precipitated DNA was analyzed by qPCR using primers specific to the p21 Cip1 locus (Table ), as indicated in the map. Fold changes are normalized to control (Ctr). TSS, transcription start site. Bars represent the means ± standard deviation from three independent experiments with measurements in triplicate in each experiment. Two‐tailed Student's t ‐test (A) and one‐way ANOVA with Bonferroni's multiple comparisons test among group (B–D); * P < 0.05, ** P < 0.01.

Article Snippet: Target sequences for nontargeting control and each gene were obtained from Mission shRNA library (Merck KGaA) (Table ). siRNA (FlexiTube siRNA) was purchased from Qiagen (Venlo, the Netherlands).

Techniques: Activity Assay, Western Blot, Quantitative RT-PCR, Transfection, Negative Control, Control, Expressing, Mutagenesis, Standard Deviation, Two Tailed Test