At 37 °C and 5% CO2, we cultivated the normal human lung epithelial cell line BEAS-2B and the human LUAD cell lines H2228, H1299, and H358 from the cell bank at the Chinese Academy of Sciences. In view of the high expression of PANX1 in H358 and H1299 cell lines as shown by WB experiments, these two cell lines were selected as the subsequent transfected cell lines. Small interfering RNA (siRNA) was obtained from OBIO Technologies to target human PANX1. After culturing H358 and H1299 cells to 50% cell density, siRNA was mixed with reduced serum medium and transfection reagent from Invitrogen and incubated for 20 min, and the siRNA complex was added to fresh medium. Six hours after transfecting the cells, the medium was replaced. Following 24–48 h of transfection, cells were gathered for validation.
Reduced serum medium
Manufactured by Thermo Fisher Scientific
Sourced in Australia, United States
Reduced serum medium is a cell culture medium formulated with a lower concentration of serum compared to standard media. It is designed to support cell growth and maintenance while minimizing the amount of serum required.
Automatically generated - may contain errors
Lab products found in correlation
Luciferase assay reagent, by Promega (2 mentions)
Transfection reagent, by Thermo Fisher Scientific (1 mentions)
Lipofectamine ltx reagent, by Thermo Fisher Scientific (1 mentions)
Pcdna3, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Lipofectamine rnaimax transfection reagent, by Thermo Fisher Scientific (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
Multimode microplate reader, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Prism 8, by GraphPad (1 mentions)
10 protocols using reduced serum medium
1
PANX1 Knockdown in Lung Cancer Cells
2
miR122-Luciferase Transfection Assay
3
Baculovirus Expression of CP and ADF Genes
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The CP and ADF genes were inserted into the vector pFAST‐T1 and pFAST‐HTB, respectively, using the primers in Table S1 . The recombinant plasmids were introduced into E. coli DH10Bac cells for transposition into the bacmid. The recombinant bacmid was used to transfect Sf9 cells. Sf9 cells were incubated in Sf‐900 III SFM Serum‐Free medium containing 5% vol/vol newborn calf serum at 27°C. The transfection of Sf9 cells with the pFAST‐T1‐CP and pFAST‐HTB‐ADF bacmids was performed with lipofectamine LTX reagent (Invitrogen). For the six‐well transfection, 1 μg bacmid and lipofectamine were diluted in 100 μl reduced‐serum medium (Invitrogen). After 5 min of incubation, lipofectamine LTX was added to the bacmid dilution and samples were incubated for another 30 min. The transfection mixture was then added to a 6‐well tissue culture plate containing Sf9 cells. A transfection mixture without any bacmid was included as the mock‐transfected control. After 5 h, each mixture was replaced with growth medium. After 2 days, the transfected Sf9 cells were fixed, permeabilized, and subsequently incubated with phalloidin and DAPI as described above. The F‐actin structure was observed with LSCM.
4
Catalase Expression via Lipofectamine 2000
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Transfection was performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) as manufacturer’s instructions. The plasmid containing the encoded sequence for catalase expression (pcDNA3, Invitrogen, USA) was amplified in competent cells (Invitrogen, Grand Island, USA). Plasmids and Lipofectamine were mixed in Reduced Serum Medium (Invitrogen), and incubated for 20 min at room temperature. For internal control, cells were transfected with empty pcDNA vector.
5
Quantifying Cell-Cell Fusion via Luciferase
6
Cell-Cell Fusion Assay for HIV Entry Inhibitors
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Cell–cell fusion
was measured as previously published22 (link) and
using cell lines obtained through the NIH AIDS Research and Reference
Reagent Program, Division of AIDS, NIAID, NIH. Target cells were TZM-bl
cells (no. 8129, contributed by J.C. Kappes, X. Wu, and Tranzyme Inc.)
expressing CD4, CCR5, and CXCR4,40 (link) and
containing an integrated reporter gene for firefly luciferase under
control of HIV-1 LTR.41 (link) Effector cells
were HL2/3 (no. 1294, contributed by B. K. Felber and G. N. Pavlakis)
which produce HXB2 Env, Tat, and Rev.42 (link) Serially diluted inhibitors were added to 96-well plates containing
25 000 TZM-bl cells per well cultured overnight. 50 000
HL2/3 cells were added per well, and fusion was allowed to proceed
for 6 h in reduced serum medium (Gibco) with a final concentration
of 1% DMSO. Luciferase expression was measured using luciferase assay
reagent (Promega) according to the manufacturer’s instructions.
Controls containing 1 μL of DMSO with and without HL2/3 cells
were measured for each compound, and experiments were performed in
triplicate.
was measured as previously published22 (link) and
using cell lines obtained through the NIH AIDS Research and Reference
Reagent Program, Division of AIDS, NIAID, NIH. Target cells were TZM-bl
cells (no. 8129, contributed by J.C. Kappes, X. Wu, and Tranzyme Inc.)
expressing CD4, CCR5, and CXCR4,40 (link) and
containing an integrated reporter gene for firefly luciferase under
control of HIV-1 LTR.41 (link) Effector cells
were HL2/3 (no. 1294, contributed by B. K. Felber and G. N. Pavlakis)
which produce HXB2 Env, Tat, and Rev.42 (link) Serially diluted inhibitors were added to 96-well plates containing
25 000 TZM-bl cells per well cultured overnight. 50 000
HL2/3 cells were added per well, and fusion was allowed to proceed
for 6 h in reduced serum medium (Gibco) with a final concentration
of 1% DMSO. Luciferase expression was measured using luciferase assay
reagent (Promega) according to the manufacturer’s instructions.
Controls containing 1 μL of DMSO with and without HL2/3 cells
were measured for each compound, and experiments were performed in
triplicate.
7
Downregulating YTHDC2 in BMSCs
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The YTHDC2 interference fragment and negative control fragment (Synbio Technologies, China) were co-transfected with Lipofectamine 2000 reagent to downregulate the target gene expression level in BMSCs according to the manufacturer's instructions. After transfection in reduced serum medium (Gibco, Australia) for 6 h, the medium was changed into OM and the BMSCs were cultured in OM for further research. The primer sequences were synthesized by the Synbio Technologies and are showing below.
| Gene | Primer type | Primer Sequence （5′-3’） |
|---|---|---|
| YTHDC2-si | Forward | CCGACUAAGUCAAUCUCUUGGUUUATT |
| Reverse | UAAACCAAGAGAUUGACUUAGUCGGTT |
8
PACE4 Knockdown Effect on Cell Growth
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Cells were plated in a 6-well plate (500,000 cells/well for LNCaP and 150,000 cells/well for JHU-LNCaP-SM). Cells were maintained in complete medium at 37 °C, 5% CO2. After 24 h, cells are transfected with siRNA specifically targeting PACE4-FL or PACE4-altCT (respectively GACAAATATTTCATATATT[dT][dT] & 5′GGAAGGAUGUACUCACAUC[dT] [dT] purchased from Sigma Aldrich). Transfections were performed using 0, 5, 10 and 25 nM in Opti-MEM, reduced serum medium (Gibco) and lipofectamine RNAiMax transfection reagent (Invitrogen) for 6 h before adding medium with FBS. After 24 h, transfection media was removed and 2 mL of complete medium is added. Following an additional 24 h, transfected cells were collected for plating in another 6-well plate as described above for colony formation assay.
9
Dextran-based siRNA Nanoparticle Synthesis
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The PD-L1 siRNA dextran NP was synthesized as previously described (20 (link)) with a few modifications. Briefly, the dextran (70 kDa) scaffold was reacted with an excess of N-(2-(bis(2-aminoethyl)amino)ethyl)-4-(4-(dimethoxymethyl)-2-methoxyphenoxy)butanamide to attach the amine groups to the dextran polymer through the acetal bonds. 1H NMR spectra indicated that the functionalized degree of glucose residues was around 0.35. Then Cy5.5 was conjugated to amine groups on the dextran platform (approximately 1 Cy5.5 molecule per dextran molecule).
This modified dextran scaffold was mixed with siRNA in reduced serum medium (ThermoFisher, Waltham, MA, USA) for cell studies, or with PBS for in vivo studies, for 20 min immediately prior to adding to cell culture or prior to injecting into mice. All the siRNA dextran NPs contained a ratio of nitrogen atoms in one dextran molecule to phosphor atoms in one siRNA molecule (N/P ratio) equal to 15.
This modified dextran scaffold was mixed with siRNA in reduced serum medium (ThermoFisher, Waltham, MA, USA) for cell studies, or with PBS for in vivo studies, for 20 min immediately prior to adding to cell culture or prior to injecting into mice. All the siRNA dextran NPs contained a ratio of nitrogen atoms in one dextran molecule to phosphor atoms in one siRNA molecule (N/P ratio) equal to 15.
10
MET Protein Interaction Monitoring
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The NanoBit Protein: Protein interactions system was obtained from Promega (N2014) and used according to the manufacturer’s protocol. Briefly, full-length MET was constructed into the provided vector to fuse LgBit and SmBit onto its C-terminus. The constructs were then transiently transfected into HEK293T cells using Lipofectamine 3000 (Invitrogen) following the manufacturer’s protocol. After 24 h, the cells were collected and seeded into 96-well plates with reduced serum medium (Thermo Fisher, 31985070). Before stimulation, Nano-Glo Live Cell Reagent provided with the kit was added and the baseline luminescence was measured using a Multimode Microplate Reader (Thermo Scientific Varioskan LUX) for 10 min. After that, purified MET404 protein (1 μg/ml) or an equivalent amount of Opti-MEM was added to each well, and the plate was gently mixed. The luminescence was measured continuously for up to 45 min. The data were recorded using SkanIt 6.1 and plotted using GraphPad Prism 8.0.
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