Cells were transfected with either PCDNA6 or PCDNA6‐Myc‐CSN6 plasmids and were selected in 8 µg mL−1 blasticidin for 2 weeks. Cells were infected by lentiviral shRNA transduction particles (Sigma, NM_0 06833) containing either shRNA or CSN6 shRNA. After infection, cells were selected with 2 µg mL−1 Puromycin for 2 weeks. For generation of COP1 overexpression stable transfectants, U2OS cells were transfected with indicated for the generation of overexpression stable transfectants.
Shrna lentiviral transduction particles
Manufactured by Merck Group
Sourced in France
ShRNA lentiviral transduction particles are a laboratory tool used for the delivery of short hairpin RNA (shRNA) into target cells. These particles contain the necessary genetic elements for efficient transduction and expression of the shRNA within the target cells, enabling researchers to study gene function and knockdown.
Automatically generated - may contain errors
Lab products found in correlation
Puromycin, by Merck Group (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Tet plko puro ires luc2 tdt, by Addgene (2 mentions)
Tet plko puro vector, by Addgene (2 mentions)
Blasticidin, by InvivoGen (1 mentions)
Combimag, by Ozyme (1 mentions)
Viromag r l beads, by Ozyme (1 mentions)
Lipornaimax, by Thermo Fisher Scientific (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
10 protocols using shrna lentiviral transduction particles
1
Stable Cell Line Generation with Overexpression and Knockdown
2
Overexpression and Knockdown of CSN6 and COP1
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Cells were transfected with either PCDNA6 or PCDNA6-Myc-CSN6 plasmids and were selected in 8 mg/ml blasticidin for 2 weeks. Cells were infected by lentiviral shRNA transduction particles (Sigma, NM_006833) containing either control shRNA or CSN6 shRNA. After infection, cells were selected with 2 mg/ml puromycin for 2 weeks. For generation of RFP-tagged-COP1 (RFP-COP1) overexpression stable transfectants, U2OS cells were transfected with RFP vector or RFP-COP1 plasmids by Electroporation (Amaxa). Forty-eight hours after transfection, cells were selected in 500 μg/ml G418 containing culture medium for 4 weeks. For generation of Myc-COP1 overexpression stable transfectants, HCT116 cells were transfected with either pCDNA6 or pCDNA6-Myc-COP1 plasmids by Electroporation (Amaxa). Forty-eight hours after transfection, cells were selected in 8 μg/ml blasticidin (Invivogen) containing culture medium for 2 weeks.
3
Stable Knockdown of OAT in MRC5 Fibroblasts
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Transient and stable transfection procedures were performed using small hairpin (sh)RNA lentiviral transduction particles according to the manufacturer’s instructions (Sigma‒Aldrich). An shRNA targeting the OAT gene (OAT shRNA [h]) and the corresponding control shRNA (h) (Sigma‒Aldrich) were used for RNA interference. For lentiviral transduction, MRC5 fibroblasts were cultured at 37 °C in 5% CO2 in 100-mm culture dishes in medium supplemented with 10% FBS (Thermo Fisher Scientific), 100 units/mL penicillin, 100 mg/mL streptomycin (Gibco), and 10 µL/mL L-glutamine (Gibco). When the cells reached 70% confluence, transduction was performed according to the manufacturer’s instructions (Sigma‒Aldrich). In brief, lentivirus expressing shRNA against human OAT (OAT knockdown) or lentivirus expressing nontargeting scrambled shRNA (scramble) was added to the cells at a multiplicity of infection (MOI) of 10. The medium was changed after 24 h. Three days after transduction, stable clones of MRC5 cells expressing OAT shRNA or scramble shRNA were selected over an additional 10 days of culture using 5 µg/mL puromycin (Sigma‒Aldrich). The medium was changed every 48 h. Gene silencing was confirmed by immunoblotting (Supplementary Fig. 1b ).
4
Inducible ERK5 Knockdown and Overexpression
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For constitutive ERK5 knockdown experiments in vitro, shRNA lentiviral transduction particles were purchased from Sigma-Aldrich to target the 3′UTR (#TRCN0000197264) or the CDS (#TRCN0000010275) of the erk5 transcript. shScr lentiviral particles (#SHC016V) were utilized as controls. To generate doxycycline-dependent inducible ERK5shRNA, oligonucleotides were designed against the GACCCACCTTTCAGCCTTA sequence at position 5639–5657 in the 3′UTR of the erk5 gene (Gene ID: 5598). Double-stranded oligonucleotides were subcloned using AgeI and EcoRI in Tet-pLKO-puro-IRES-Luc2=tdT, a lentiviral transfer plasmid created by inserting a Luc2=tdT fragment (Addgene #32904) into the Tet-pLKO-puro vector (Addgene #21915). Sequences for shERK5i are available in Supplementary Table S1 . To construct doxycycline-dependent inducible expression systems, F-ERK5(WT) or D200A mutant cDNAs were subcloned using PacI and NheI into the bicistronic lentiviral expression vector pCHD-TRE3GS-MCS-EF1a-iRFP720 (a gift from Stuart Cain, University of Manchester, in which target cDNA expression is driven by the TRE3GS promoter and iRFP720 expression is driven by the EF-1α core promoter). Sequences for PCR amplification are available in Supplementary Table S1 .
5
Lentiviral Transduction and Sp1 Knockdown
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shRNA lentiviral transduction particles (Sigma-Aldrich) were used and shRNA transduction was performed with the MagnetoFection-ViroMag R/L (OZ Biosciences, Marseille, France) as previously described. Sp1 shRNA (NM_138473.2-5s21c1) (Sigma-Aldrich) or control shRNA (Sigma-Aldrich) were designed by MISSION®. To increase the transduction efficiency, 1 mL of RPMI8226 cell suspension (105 cells) was mixed with 20 μL CombiMag® (OZ Biosciences) and incubated for 15 minutes. Then, the cells were distributed to 96-well plates (100 μL/well) and placed for 15 minutes on a magnetic plate. To prepare lentiviral transduction particles, 1 multiplicity of infection (MOI) of lentiviral particles were added to 2 μL of ViroMag R/L beads (OZ Biosciences), and incubated for 15 minutes at room temperature. Then the virus particles/ViroMag R/L were added to each well, and the cells were incubated on the magnet plate for 60 minutes at room temperature. The culture plates were removed from the magnetic plate and the cells were cultured at 37°C with 5% CO2 for 24 hours. To establish stable Sp1 knockdown cells, clones were selected by treatment with puromycin (Sigma) at 5 μg/mL.
6
Inducible ERK5 Knockdown and Overexpression
7
Lentiviral Knockdown of FASN Gene
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Transient and stable transfection procedures were performed with small hairpin (sh) RNA Lentiviral Transduction Particles according to the manufacturer’s instructions (Sigma-Aldrich). The shRNA against the FASN gene (FASN shRNA) and the corresponding control shRNA (Sigma-Aldrich) were used for RNA interference. For lentiviral transduction, A549 cells were cultured at 37 °C under 5% CO2 in 100 mm culture dishes containing 10% FBS (Gibco), 100 units/mL penicillin, 100 mg/mL streptomycin (Gibco), and 10 μL/mL l -glutamine (Gibco). When cells reached 70% confluence, transduction was performed according to the manufacturer’s instructions (Sigma-Aldrich). Then, human-FASN shRNA-expressing lentivirus (FASN knockdown) or nontargeting shRNA-expressing scrambled RNA sequence (scramble) was added to the cells at a multiplicity of infection (MOI) of 5. The medium was changed after 24 h. Three days after transduction, stable clones expressing FASN shRNA and scramble RNA were selected over a further 10 days using 5 μg/mL puromycin (Sigma-Aldrich). The medium was changed every 48 h. Gene silencing was confirmed by Western blotting and reverse transcription polymerase chain reaction (RT-PCR).
8
Lentiviral shRNA Knockdown in Cells
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MISSION shRNA lentiviral transduction particles were purchased from Sigma-Aldrich. Cells were infected at MOI = 1 for 2–3 days and selected with 5 μg/ml Puromycin for 3–4 days or until all the uninfected cells were killed. Puromycin resistant cells were cultured for at least two weeks before use. shRNA clones used are the following:
| shRNA | Product no. | Clone no. |
|---|---|---|
| shSNAI2-284362 | SHCLNV-NM_003068 | TRCN0000284362 |
| shSNAI2-271239 | SHCLNV-NM_003068 | TRCN0000271239 |
| shPARP1-7928 | SHCLNV-NM_001618 | TRCN0000007928 |
| shPARP1-7929 | SHCLNV-NM_001618 | TRCN0000007929 |
| shPARP1-7930 | SHCLNV-NM_001618 | TRCN0000007930 |
| shPARP1-7931 | SHCLNV-NM_001618 | TRCN0000007931 |
| shPARP1-7932 | SHCLNV-NM_001618 | TRCN0000007932 |
| shCHD1L-13469 | SHCLNV-NM_004284 | TRCN0000013469 |
| shCHD1L-280395 | SHCLNV-NM_004284 | TRCN0000280395 |
| shNon | SHC002V |
9
Characterization of Pancreatic and Breast Cancer Cell Lines
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The human pancreatic adenocarcinoma BxPC-3 cells, authenticated using short tandem repeat (STR) profiling (ATCC), the pancreatic cancer primary culture PDAC001T and Human breast cancer cells (SUM-149) were cultured as previously published (24, (link)49, (link)50) (link). E-cadherin was stably knockdown in the BxPC-3 cell line using shRNA lentiviral transduction particles (Sigma) as previously described( 24). E-cadherin extinction was checked every 3 weeks by Western Blot analysis.
Antibodies and reagents. Working dilutions and information of commercial antibodies and chemical reagents used in this study are listed in Supplemental Table 1.
Reverse siRNA transfection. For protein knockdown, reverse transfections were performed in 6 well plates. Cells were seeded directly with the siRNA/Transfection mix: 3µl of LipoRNAiMax (Lifetechnologies), 500µL of OptiMEM (Gibco, Lifetechnologies), 25 or 50nM of indicated siRNA and 2.5ml of RPMI/10% FCS medium. siRNA used for these studies are listed in Supplemental Table 2. When required, transfected cells were detached and seeded on FITC-gelatin coverslips, 24 or 48h after treatment.
Antibodies and reagents. Working dilutions and information of commercial antibodies and chemical reagents used in this study are listed in Supplemental Table 1.
Reverse siRNA transfection. For protein knockdown, reverse transfections were performed in 6 well plates. Cells were seeded directly with the siRNA/Transfection mix: 3µl of LipoRNAiMax (Lifetechnologies), 500µL of OptiMEM (Gibco, Lifetechnologies), 25 or 50nM of indicated siRNA and 2.5ml of RPMI/10% FCS medium. siRNA used for these studies are listed in Supplemental Table 2. When required, transfected cells were detached and seeded on FITC-gelatin coverslips, 24 or 48h after treatment.
10
Calcium Signaling in Cell Lines
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Cell Culture and Transfection HeLa, HEK293T, and COS7 cells were propagated and maintained as American Type Culture Collection (ATCC) recommended. Several TMCO1 knocking-down cell lines were generated by infection with small hairpin RNA (shRNA) lentiviral transduction particles produced by Sigma and maintained in DMEM containing puromycin with 10% FBS.
[Ca 2+ ]cyto and [Ca 2+ ] ER Measurements Cytosolic Ca 2+ concentration was measured by Fura-2 or Fluo4 as described previously (Tang et al., 2003) , and [Ca 2+ ] ER measurements were done with D1ER probe as described before (Palmer et al., 2004) . For more details, see the Supplemental Experimental Procedures.
[Ca 2+ ]cyto and [Ca 2+ ] ER Measurements Cytosolic Ca 2+ concentration was measured by Fura-2 or Fluo4 as described previously (Tang et al., 2003) , and [Ca 2+ ] ER measurements were done with D1ER probe as described before (Palmer et al., 2004) . For more details, see the Supplemental Experimental Procedures.
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