Cell culture and differentiation. Human teratocarcinoma NTERA-D1 cells from the (referred to as NT2 thereinafter) American Type Culture Collection (ATCC®, CRL-1973TM) were maintained in complete medium consisting of Dulbecco’s Modified Eagle Medium (DMEM®, ATCC® 30-2002™) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Sigma-Aldrich, St Louis, MO, USA) and antibiotics (100 U/ mL penicillin and 100 µg/mL streptomycin) (Gibco, Life Technologies S.A., Madrid Spain) at 37°C under a humidified atmosphere containing 5% CO2. NT2 progenitors were treated as previously described for neuronal differentiation with all-trans retinoic acid (RA, Sigma-Aldrich) (5) (3) and cytosine-β-D-arabinofuranoside (AraC) (6) with slight modifications and improvements for use in our laboratory (5) (36).
Rat PC12 cells (ATCC®, CRL-1721) of no more than 8 passages were grown in complete PC12 medium consisting of DMEM supplemented with 10% heat-inactivated horse serum (Thermo Fisher Scientific), 5% FBS and antibiotics at 37°C under 5% CO2. NGF-induced neuronal was carried out on cells that had reached ~ 80% confluency. Briefly, complete PC12 medium was replaced with PC12 differentiation medium consisting of DMEM supplemented with 1% heat-inactivated horse serum, 0.1 µg/ml recombinant β-NGF (Bio-Techne, 256-GF-100/CF, Minneapolis USA ) and antibiotics and incubated for 72 h. Cells treated identically but incubated
the DNA sequences encoding the human mRNA splice variants of PLCβ1a and PLCβ1b and their corresponding bipartite nuclear localization signal (NLS) mutants PLCβ1a-M2b and PLCβ1b-M2b into the mammalian expression bicistronic plasmid pIRES-EGFP-puro (Addgene, #45567, Cambridge, MA, USA) was performed from the commercial plasmids pCMV6-XL6-PLCb1a (#SC126664, OriGene Technologies, Inc, Rockville, USA. USA) and pCMV6-XL6-PLCb1a (#SC309945, OriGene Technologies, Inc.) using molecular biology approaches including conventional PCR, overlap extension PCR and annealing of in P12 differentiation medium without NGF were used as control.
Molecular cloning. Five double-stranded DNA sequences coding for short-hairpin RNAs (shRNAs), including four encoding siRNAs targeting the human PLCB1 transcript (shRNA-PLCβ1–87, 89, 97, 99) and one non-target scrambled-base DNA (shRNA-Scrmbl-01), were inserted into the BglII/HindIII cloning site of pSuperior.gfp/neo mammalian expression vector (Oligoengine, #VEC-IND-0007, Seattle, WA, USA) downstream the RNA polymerase III H1-RNA gene promoter. Cloning of custom-designed complementary oligonucleotide pairs followed by restriction/ligation-based cloning. Before being subcloned into the multiple cloning site (MCS) of pCDNA3 plasmid, the original inserts containing the open reading frames (ORFs) for PLCβ1a and PLCβ1b in the pCMV6-XL6 vector were modified [i] to remove long fragments corresponding to the 5' and 3' UTR regions of the mature mRNAs, [ii] to replace the native translation initiation site with the Kozak consensus sequence (GCC ACC ATG G) and [iii] to add AgeI and NotI unique restriction sites and at the 5' and 3' ends, respectively, for subsequent subcloning. In addition, site-directed mutagenesis by PCR was used to reverse a C > T single nucleotide discrepancy (TCA to TTA) detected in commercial plasmids and leading to a S1156L substitution in the protein sequence of human PLCβ1a and PLCβ1b (NCB1 accession, NP_056007.1 and NP_877398.1, respectively). The pcDNA-PLCβ1a and pcDNA-PLCβ1b constructs thus obtained were used for overlapping PCR mutation of the bipartite nuclear localization signal spanning residues 1055–1072 of both splice variants (KKLKEICEKEKKELKKKM), thus generating the constructs pcDNA-PLCβ1a-M2b and pcDNA-PLCβ1b-M2b encoding the PLCβ1a-M2b and PLCβ1b-M2b mutant variants in which three lysine residues of the NLS were replaced by isoleucine (K1056I, K1063I and K1070I), preventing their nuclear localization (26, 27, 37). Final PLCβ1a, PLCβ1b, PLCβ1a-M2b and PLCβ1b-M2b constructs were subcloned by restriction/ligation into the MCS downstream of the cytomegalovirus promoter of pIRES-EGFP-puro.
Transfection of Plasmids and siRNAs. NT2 cells at ∼70% confluence were transfected with DNA inserts of PLCβ1 constructs into pDCNA 3.0 or in the pIRES-EGFP-puro (Addgene, #45567, Cambridge, MA, USA) which allows the synthesis of two proteins from a single bicistronic mRNA translated through the cap-independent Internal Ribosome Entry Site (IRES)-mediated mechanism for the simultaneous and separate expression. Transfection with PLCβ1 shRNAs cloned downstream of the H1 promoter in the pSuperior.gfp/neo plasmid (encoding eGFP under the control of a separate PGK promoter) was performed identically, except that a cocktail prepared by mixing equimolar concentrations four different custom-designed shRNAs targeting different regions of the human PLCβ1 transcript was used. Transfection was carried out with Lipofectamine® 2000 (Invitrogen™) in Opti-MEM™ I Reduced Serum Medium (Thermo Fisher Scientific) without antibiotics according to the manufacturer’s instructions, using a ratio of 2 µl Lipofectamine to 1 µg DNA, using 0.5, 1 and 2.5 µg for 24-, 12- and 6-well plates, respectively. 6 h after transfection, the medium was replaced with complete medium.
PC12 cells at ∼70% were transfected with mouse eGFP-PLCβ1a cloned in pCDNA 2.0 plasmid (a gift from Dr. Catherine Berlot) using Lipofectamine™ 3000 (Invitrogen™) as described for NT2 cells, and 6 h after transfection, the medium was replaced. In all cases, the corresponding empty plasmid (mock) was used as control.
For small interfering (siRNA)-mediated gene knockdown in human NT2 and rat PC12 cells, we used ON-TARGET plus SMART pool siRNAs, which consist of species-specific mixture of four siRNAs targeting different regions of the human (Dharmacon™, Cat. L-010280-00-0010) or rat (Dharmacon™, Cat. L-092936-02-0010) PLCβ1 mRNA. For control experiments, we used ON-TARGETplus Non-targeting Control siRNA Pool (Dharmacon™, Cat. D-001810-10-20), which consists of four small RNAs designed for minimal targeting of human, mouse or rat genes and modified to reduce potential off-targets. Transfection was done on cells at ∼70% using DharmaFECT 1 Transfection Reagent (Dharmacon™, Cat. T-2001-02) in antibiotic and serum-free medium at a ratio of 1 µL DharmaFECT 1 to 2 nmol siRNA, using DharmaFECT 1 The transfection mix was prepared according to the manufacturer’s protocol, using a ratio using a ratio of 2.5 µL of DharmaFECT 1 Reagent for 5 nmol siRNA. 6 h after transfection, the medium was replaced with complete medium and the cells were allowed to grow until harvested or fixed for immunofluorescence staining and microscope analysis.
Western Blotting NT2 cells were pelleted under different conditions as indicated in the text and lysed in homogeneization buffer (10 mM Tris-HCl buffer, pH 7.4, 2 mM MgCl2, and 0.32 M sucrose) containing protease inhibitors (0.5 mM iodoacetamide and 1 mM phenylmethylsulfonyl fluoride, PMSF). Depending on the antigen to be analyzed, different amounts of denatured lysates (5–30 µg) were loaded, resolved by SDS-polyacrylamide (SDS-PAGE) gels and transferred to polyvinylidene fluoride membranes (Bio-Rad, Madrid, Spain), and specific protein bands were detected using conventional inmunoblot protocols. Primary and secondary antibodies are described in Table S1 and Table S2, respectively.
Western blotting to detect proteins in PC12 cells was carried out by lysing cells in ice-cold NP40 buffer (pH = 8.0, 30 ml 5 M NaCl, 100 ml 10% NP40, 50 ml 1 M Tris base, 820 ml diH2O) (pH = 8.0, 0.15 M NaCl, 1% NP40, 0.05 M Tris base) for ~ 10 min at room temperature. To prevent protein degradation, Pierce Protease Inhibitor Mini Tablets were added into NP40 lysis buffer (1 tablet per 10 ml solution, Thermo Fisher Scientific), and the mixture was transferred into 1.5 ml Eppendorf tubes and incubated at 4 ℃ for 30 min on a shaker. Tubes were centrifuged at 4 ℃, 12,000 rpm (c.a. 17,000 RCM, VWR™ Galaxy 14D Digital Microcentrifuges) for 20 min. After centrifugation, supernatant/lysate was transferred into new 1.5 ml Eppendorf tubes. Bradford protein assays were use to approximate protein concentrations of each lysate. Denatured lysates were mixed with loading buffer at 70 ℃ for 10 min. After denaturation, loaded lysates, SeeBlue™ Plus2 Pre-stained Protein Standard, and MagicMark™ XP Western Protein Standard (Thermo Fisher Scientific) into 4–15% Mini-PROTEAN® TGX Stain-Free™ Protein Gels, 10 well, 50 µl (Bio-Rad). Both gel electrophoresis and wet-transfer were performed by employing Bio-Rad’s Mini-PROTEAN Tetra Cell System and normalized to total protein (protocol provided by Azure Biosystems). Electrophoresis was performed at room temperature, 100 V for around 2 h. After electrophoresis, gels were imaged (Azure Biosystem C600), at 302 nm UV override for 5 min to stimulate trihalo compounds reaction with tryptophan residues to produce fluorescence so that a total protein gel image can be captured. After imaging, slow wet-transfer (4 ℃, 30 V for 999 min) was performed to transfer proteins from gels to nitrocellulose membranes for blotting. After transfer, membranes were blocked by buffer (TBST buffer with 5% (m/v) nonfat milk powder) for 1 h at 4 ℃. For blotting, indirect ECL method was performed for PLCβ1 probing: the primary antibody used is Anti-PLCβ1 Antibody (D-8), and the secondary antibody is m-IgGκ BP-HRP (Santa Cruz Biotechnologies, sc-5291 and sc-516102). EGR1 was probed by employing direct ECL method: the antibody used is Anti-Egr-1 Antibody (B-6) HRP (Santa Cruz Biotechnologies sc-515830 HRP). Anti-PLCβ1 Antibody (D-8) were removed from membranes by incubating membranes with Restore™ PLUS Western Blot Stripping Buffer (Thermo Fisher Scientific 46430) for 10 min at 37 ℃ after PLCβ1 probing and before EGR1 probing. Data and images were analyzed and plotted on AzureSpot Analysis Software and GraphPad Prism 9 software.
TRBP Immunoprecipitation. Passage 9 PC12 cells were cultured in 20 Nunc™ EasYDish™ Dishes (145 cm2, Thermo Fisher Scientific) until around 70% confluence. 10 of the 20 dishes were transfected with eGFP-PLCβ1 using Lipofectamine™ 3000 Transfection Reagent (Thermo Fisher Scientific) where the sequence was verified by Plasmidsaurus, Inc.). Cell were incubated with the transfection solution in an antibiotic-free culture medium for c.a. 60 h. Control dishes used the same antibiotic-free culture medium. After incubation, cells were lysed with NP40 lysis buffer (pH = 8.0, 30 ml 5 M NaCl, 100 ml 10% NP40, 50 ml 1 M Tris base, 820 ml diH2O), loaded one tablet of Pierce™ Protease Inhibitor Mini Tablets, EDTA-free (Thermo Fisher Scientific) per 10 mL liquid.
For immunoprecipitation, Pierce™ Gentle Ag/Ab Binding Buffer (pH = 8.0, Thermo Fisher Scientific) was always used for the wash and binding steps. TRBP Monoclonal Antibody (OTI2C12, Invitrogen™) was combined with Dynabeads™ Protein G (Invitrogen™). Two cell lysates were incubated with the antibody-conjugated beads separately on a 3D shaker at room temperature for 1 h to let TRBP in cell lysates bind with beads and then isolated using a magnetic stand and washed thoroughly. Beads were incubated with Pierce™ Gentle Ag/Ab Elution Buffer (pH = 6.6, Thermo Fisher Scientific) at room temperature for 15 min on a 3D shaker and the eluates collected. Used spin filters were used to replace the solvent of these protein solutions with 1xTBS buffer, as well as increase the protein concentrations. Protein concentrations of two samples were determined by performing a BCA assay.
For gel electrophoresis, protein samples were mixed with sample loading buffer in appropriate ratio, then denatured at 70 ℃ for 10 min. After denaturation, loaded protein samples, Novex™ Sharp Pre-stained Protein Standard, and MagicMark™ XP Western Protein Standard (Thermo Fisher Scientific) into 4–15% Mini-PROTEAN® TGX Stain-Free™ Protein Gels, 10 well, 50 µl (Bio-Rad). Both gel electrophoresis and wet-transfer were performed by employing Bio-Rad’s Mini-PROTEAN Tetra Cell System. Electrophoresis was performed at room temperature, 100 V for around 90 min. After electrophoresis, gels were imaged (Azure Biosystem C600), at 302 nm UV override for 5 min to stimulate trihalo compounds reaction with tryptophan residues to produce fluorescence so that a total protein gel image can be captured. After imaging, slow wet-transfer (4 ℃, 30 V for 999 min) was performed to transfer proteins from gels to nitrocellulose membranes for blotting. After transfer, membranes were blocked by nonfat milk blocking buffer (TBST buffer with 5% (m/v) nonfat milk powder, Santa Cruz Biotechnologies, sc-2324) for 1 h at 4 ℃ on a 2D shaker. Then discarded the blocking buffer and rinsed membranes with TBST buffer. The membranes were ready for blotting.. Both primary and secondary antibodies (see Tables S1 and S2) were dissolved in the 5% nonfat milk (Blotto, non-fat dry milk, sc-2324, Santa Cruz Biotechnologies) blocking buffer. After imaging, ladder areas were cut off from membranes, and antibodies from the rest parts of membranes were removed by incubating membranes with Restore™ PLUS Western Blot Stripping Buffer (Thermo Fisher Scientific, 46430) for 10 min at room temperature. Performed imaging again on the stripped membranes to make sure no bands showed up except ladders. After stripping, we probed EGR1 using direct ECL. Anti-Egr-1 Antibody (B-6) HRP (Santa Cruz Biotechnologies sc-515830 HRP) was dissolved in BSA blocking buffer (TBST buffer with 5% (m/v) BSA, fraction V, MP Biomedicals LLC Cat#820451) in 1:500 ratio.
Data were analyzed using ImageJ. TRBP bands were selected by same rectangle ROI to determine the gross band mean gray value, and backgrounds of each lane. The net intensity of each TRBP band was the difference of gross band mean gray value and background mean gray value (Eq. 1). For EGR1, bands were similarly selected and analyzed. After obtaining the net intensities of TRBP and EGR1 bands, the relative expression level of EGR1 was calculated by dividing each EGR1 net intensity with their corresponding TRBP net intensity (Eq. 2) for the TRBP band from the same lane. Control samples were used to calculate the normalized fold expression level by dividing relative EGR1 expression level of each data point with the mean value of relative EGR1 expression level of all lanes from control sample (Eq. 3). Normalized fold expression levels of EGR1 in two different treatments (Control and PLCβ1+) were analyzed and plotted by using GraphPad Prism 10 software. Unpaired t test comparison was performed to show the significance of differences: ns (P > 0.05), * (P ≤ 0.05), ** (P ≤ 0.01), *** (P ≤ 0.001), and **** (P ≤ 0.0001).
\(\text{N}\text{e}\text{t} \text{I}\text{n}\text{t}\text{e}\text{n}\text{s}\text{i}\text{t}\text{y}=\) Gross Band Mean Gray Value - Background Mean Gray Value (Eq. 1)
\(\text{R}\text{e}\text{l}\text{a}\text{t}\text{i}\text{v}\text{e} \text{E}\text{G}\text{R}1 \text{E}\text{x}\text{p}\text{r}\text{e}\text{s}\text{s}\text{i}\text{o}\text{n} \text{L}\text{e}\text{v}\text{e}\text{l}= \frac{\text{E}\text{G}\text{R}1 \text{N}\text{e}\text{t} \text{I}\text{n}\text{t}\text{e}\text{n}\text{s}\text{i}\text{t}\text{y}}{\text{T}\text{R}\text{B}\text{P} \text{N}\text{e}\text{t} \text{I}\text{n}\text{t}\text{e}\text{n}\text{s}\text{i}\text{t}\text{y}}\) (Eq. 2)
\(\text{N}\text{o}\text{r}\text{m}\text{a}\text{l}\text{i}\text{z}\text{e}\text{d} \text{F}\text{o}\text{l}\text{d} \text{E}\text{x}\text{p}\text{r}\text{e}\text{s}\text{s}\text{i}\text{o}\text{n} \text{L}\text{e}\text{v}\text{e}\text{l}= \frac{\text{R}\text{e}\text{l}\text{a}\text{t}\text{i}\text{v}\text{e} \text{E}\text{G}\text{R}1 \text{E}\text{x}\text{p}\text{r}\text{e}\text{s}\text{s}\text{i}\text{o}\text{n} \text{L}\text{e}\text{v}\text{e}\text{l}}{\text{m}\text{e}\text{a}\text{n} \text{v}\text{a}\text{l}\text{u}\text{e} \text{o}\text{f} \text{r}\text{e}\text{l}\text{a}\text{t}\text{i}\text{v}\text{e} \text{E}\text{G}\text{R}1 \text{e}\text{x}\text{p}\text{r}\text{e}\text{s}\text{s}\text{i}\text{o}\text{n} \text{l}\text{e}\text{v}\text{e}\text{l} \text{o}\text{f} \text{a}\text{l}\text{l} \text{l}\text{a}\text{n}\text{e}\text{s} \text{f}\text{r}\text{o}\text{m} \text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l} \text{s}\text{a}\text{m}\text{p}\text{l}\text{e}}\) (Eq. 3)
Immunofluorescence
NT2 cells were fixed in buffered 4% paraformaldehyde for 4 min, both at 20–25°C. After blocking for 1 h with gelatin-histology buffer (0.1 M PBS, pH 7.4, containing 0.22% gelatin (Panreac, Barcelona, Spain), 0.05% saponin (Sigma-Aldrich), and 1% serum albumin bovine (BSA, sigma-Aldrich), cells were incubated with primary antibodies (Table S1, for details) at 4°C overnight. Then, the appropriate fluorescent dye-conjugated secondary antibodies (Table S2 for details) were applied and nuclei were counterstained with Hoechst 33,342 (Sigma-Aldrich, diluted to 0.1 µg/mL in gelatin histology buffer).
PC12 cells from different dishes were transfected with PLCβ1-eGFP plasmid, pcDNA PLCβ1 plasmid, rat PLCβ1 siRNA, and/or treated with NGF. Control dishes included Ab-free medium control cells, PC12 differentiation control cells, or complete PC12 medium. After specific times, cells were fixed by 3.7% formaldehyde/DPBS solution (Sigma Aldrich and Thermo Fisher Scientific) for 10 min at room temperature. Formaldehyde was discarded, and cell dishes were rinsed by DPBS several times. Fixed cell dishes were rinsed by MSM pipes (0.4% (v/v) Triton X-100 in DPBS, Sigma Aldrich and Thermo Fisher Scientific) 3 times, 10 min each, then incubated each dish with blocking buffer (5% (v/v) goat serum, 1% (m/v) BSA, and 50 mM glycine) for 30 min at room temperature to prevent unspecific binding. Cells were then stained by DAPI (Roche Life Science) by following its product protocol. For PLCβ1-EGR1 localization study, cells transfected with rat PLCβ1 siRNA or induced by NGF, and control cells were immunostained by primary antibody Anti-PLCβ1 Antibody (D-8) (1:50 dilution, Santa Cruz Biotechnologies, sc-5291), and then secondary antibody Goat anti-Mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ Plus 488 (1:200 dilution, Thermo Fisher Scientific, # A32723). After immunostaining, rinsed cell dishes with DPBS several times to completely remove unbound antibodies to prevent cross-staining. Then cells were immunostained by Anti-Egr-1 Antibody (B-6) Alexa Fluor® 647 (1:50 dilution, Santa Cruz Biotechnologies, sc-515830 AF647). Unbound antibodies were washed away by DPBS. For ERK 1/2 localization study, cells transfected by pcDNA PLCβ1 or rat PLCβ1 siRNA, and control cells were immunostained by primary antibody ERK 1/2 Antibody (H-72) (1:200 dilution, Santa Cruz Biotechnologies, sc-292838), and then secondary antibody Chicken anti-Rabbit IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488 (1:400 dilution, Thermo Fisher Scientific, # A-21441).
Cells were imaged on a Nikon Eclipse TI-FLBW-E using 60× objective and NIS-Elements AR software. Images were collected at DAPI, FITC (for eGFP and Alexa 488), and TRITC (for Alexa 647) channels for every captured cell. Cell images were analyzed on ImageJ. The freehand selection tool was used to circle cells and their nuclei. Each cell was circled on the merged images, and their nucleus were circled in blue channel images. The mean gray values in every cell and nucleus were measured in red channel images and green channel images. Nuclear Intensity/Cellular Intensity values were calculated by using Eq. (4) to quantify the nuclear aggregations of target proteins in cells which were treated with different conditions. Data from images were analyzed and plotted on GraphPad Prism 9 software.
\(Nuclear Intensity/Cellular Intensity=\frac{Mean Gray Value of Target Protein in Nuclei}{Mean Gray Value of Target Protein in Cell}\) (Eq. 4)
Statistical analysis. Results were statistically analyzed in GraphPad Prism (version 5.0, GraphPad Software Inc., San Diego, CA) and analyzed using the packages as indicated in the figure legends.