Journal: EMBO Molecular Medicine

Article Title: Cited4 is a sex‐biased mediator of the antidiabetic glitazone response in adipocyte progenitors

doi: 10.15252/emmm.201708613

Figure Lengend Snippet: A mRNA expression in FACS‐isolated Lin(Ter119/CD31/CD45) − Sca1 + progenitor cells from female mouse subcutaneous fat, differentiated in the presence of 1 μM cPGI 2 or vehicle for the indicated time, as determined by expression profiling ( n = 3, E‐MTAB‐3693). **** P = 3 × 10 −6 (Day 2), **** P = 4 × 10 −7 (Day 4), **** P = 1 × 10 −6 (Day 6) in 2 × 2 ANOVA with Bonferroni's posttests (cPGI 2 vs. vehicle). B mRNA expression in MACS‐isolated Lin − Sca1 + progenitor cells from female mouse subcutaneous fat, differentiated in the presence of 100 nM Rosi or vehicle for the indicated time, as determined by qRT–PCR ( n = 4). **** P = 1 × 10 −10 (Days 1 and 2), ** P = 0.001, in 2 × 2 ANOVA with Bonferroni's posttests (Rosi vs. vehicle). C mRNA expression in female Lin − Sca1 + cells, differentiated in the presence of 100 nM Rosi or vehicle for 8 days, as determined by qRT–PCR ( n = 3). t ‐test Cited4 −/− vs. Cited4 +/+ (Rosi) * P = 0.013 ( Ucp1 ), ** P = 0.004 ( Cpt1b ), * P = 0.026 ( Dio2 ). D–F mRNA expression in primary SVF cells from human subcutaneous fat, differentiated in the presence of 100 nM Rosi (D) or vehicle (D–F), as determined by qRT–PCR at the indicated time points ( n = 5 patients). ♀/♂ represents individual data. (D) **** P = 3 × 10 −5 (Day 2), **** P = 3 × 10 −6 (Day 6), **** P = 4 × 10 −9 (Day 10), **** P = 3 × 10 −7 (Day 14), in 2 × 2 ANOVA with Bonferroni's posttests (Rosi vs. vehicle). (E, F) Pearson correlation coefficient ( r ) and P ‐value are shown. G mRNA expression in primary SVF cells from human female subcutaneous fat transfected with the indicated siRNA prior to differentiation in the presence of 100 nM Rosi for 9 days, as determined by qRT–PCR ( n = 3). *** P = 0.0002 ( CITED4 ), ** P = 0.002 ( UCP1 ), * P = 0.02 ( UCP1 ), * P = 0.035/0.026 ( PPARG ), *** P = 0.0006 ( SLC2A4 ), ** P = 0.002 ( ADIPOQ ) in one‐way ANOVA with Tukey's posttests (vs. siCtrl ). Data information: Data are presented as mean ± SEM except for (D) ♀/♂, (E and F) individual data.

Article Snippet: For the transfection of primary Lin − Sca1 + cells from Cited4 F/F mice with StemMACSTM Cre recombinase (130‐101‐113; Miltenyi Biotec) or StemMACSTM Nuclear eGFP messenger RNA (mRNA) (130‐101‐119; Miltenyi Biotec), a mixture of Lipofectamine ® RNAiMAX transfection reagent (2 μl/ml) and Opti‐MEM medium was combined with Opti‐MEM containing the respective mRNA (500 ng/ml final concentration).

Techniques: Expressing, Isolation, Quantitative RT-PCR, Transfection

Journal: EMBO Molecular Medicine

Article Title: Cited4 is a sex‐biased mediator of the antidiabetic glitazone response in adipocyte progenitors

doi: 10.15252/emmm.201708613

Figure Lengend Snippet: A mRNA expression in C3H10T1/2 cells, differentiated in the presence of 1 μM Rosi or vehicle for the indicated time, as determined by qRT–PCR (day 0, n = 3; day 4, n = 4; day 6, Ctrl, n = 2, Rosi, n = 4; day 10, n = 4). *** P = 0.001, ** P = 0.007 (Day 6), ** P = 0.005 (Day 10), in 2 × 2 ANOVA with Bonferroni's posttests (Rosi vs. vehicle). B mRNA expression in 3T3‐L1 cells, differentiated in the presence of 1 μM Rosi or vehicle for the indicated time, as determined by qRT–PCR ( n = 3). 2 × 2 ANOVA with Bonferroni's posttests, P > 0.05 (Rosi vs. vehicle). C mRNA expression in female Lin − Sca1 + cells, differentiated in the presence of 0.1 or 1 μM pioglitazone (Pio) or vehicle for 8 days, as determined by qRT–PCR ( n = 4 for Day 2, n = 4/2/6 for Day 8). *** P = 1*10 −10 , ** P = 0.003, in 2 × 2 ANOVA with Bonferroni's posttests (Pio vs. vehicle). D mRNA expression in female Lin − Sca1 + cells, differentiated in the presence of the indicated substances for 8 days, as determined by qRT–PCR ( n = 3). ** P = 0.0035, *** P = 4 × 10 −10 , *** P = 7 × 10 −10 ( Ucp1 ), * P = 0.036, *** P = 0.0002, *** P = 1 × 10 −10 , *** P = 1 × 10 −10 ( Cpt1b ), *** P = 2 × 10 −5 ( Cidea ), *** P = 1 × 10 −6 , *** P = 4 × 10 −9 , *** P = 1 × 10 −8 , ( Elovl3 ), *** P = 9 × 10 −8 , *** P = 2 × 10 −7 ( Cox7a1 ), *** P = 0.0002, *** P = 1 × 10 −8 , *** P = 4 × 10 −5 , *** P = 0.0002 ( Cox8b ), * P = 0.032, ** P = 0.006, *** P = 0.0007 ( Dio2 ), *** P = 1 × 10 −6 , *** P = 2 × 10 −8 ( Cyc1 ), *** P = 8 × 10 −5 , *** P = 3 × 10 −6 ( Ndufb3 ) in 2 × 2 ANOVA with Holm–Sidak posttests ( Cited4 −/− vs. Cited4 +/+ ). E mRNA expression in male Lin − Sca1 + progenitor cells, differentiated in the presence of 100 nM Rosi or vehicle for the indicated time, as determined by qRT–PCR ( n = 3). **** P = 1 × 10 −10 (Days 2 and 4), **** P = 4 × 10 −8 (Day 8) in 2 × 2 ANOVA with Bonferroni's posttests (Rosi vs. vehicle). F mRNA expression in male Lin − Sca1 + cells, differentiated in the presence of 100 nM Rosi or vehicle for 8 days, as determined by qRT–PCR ( n = 3, t ‐test). Data information: Data are presented as mean ± SEM except for (C) (individual data).

Article Snippet: For the transfection of primary Lin − Sca1 + cells from Cited4 F/F mice with StemMACSTM Cre recombinase (130‐101‐113; Miltenyi Biotec) or StemMACSTM Nuclear eGFP messenger RNA (mRNA) (130‐101‐119; Miltenyi Biotec), a mixture of Lipofectamine ® RNAiMAX transfection reagent (2 μl/ml) and Opti‐MEM medium was combined with Opti‐MEM containing the respective mRNA (500 ng/ml final concentration).

Techniques: Expressing, Quantitative RT-PCR

Journal: EMBO Molecular Medicine

Article Title: Cited4 is a sex‐biased mediator of the antidiabetic glitazone response in adipocyte progenitors

doi: 10.15252/emmm.201708613

Figure Lengend Snippet: A Phase contrast microscopy of primary SVF cells from human subcutaneous fat, differentiated in the presence of 100 nM Rosi or vehicle for 14 days (representative of n = 5 patients). Scale bar is 100 μm. B, C mRNA expression in primary SVF cells from human subcutaneous fat, differentiated in the presence of 100 nM Rosi or vehicle, as determined by qRT–PCR ( n = 5 patients). (B) **** P = 1 × 10 −10 (Day 2), **** P = 5 × 10 −8 (Day 6), **** P = 1 × 10 −9 (Days 10 and 14), (C) **** P = 7 × 10 −7 (Day 6), **** P = 1 × 10 −9 (Days 10 and 14) in 2 × 2 ANOVA with Bonferroni's posttests (Rosi vs. vehicle). D mRNA expression in primary SVF cells from human male subcutaneous fat transfected with the indicated siRNA prior to differentiation in the presence of 100 nM Rosi for 9 days, as determined by qRT–PCR ( n = 3). *** P = 0.0002 ( siCITED4.1 CITED4 ), *** P = 0.0003 ( siCITED4.2 CITED4 ), ** P = 0.005 ( UCP1 ), * P = 0.01 ( CIDEA ), *** P = 0.0006 ( CITED4.1 CPT1B ), ** P = 0.007 ( CPT1B ), *** P = 0.0006 ( CITED4.1 SLC2A4 ),** P = 0.009 ( ADIPOQ ) in ANOVA with Tukey's posttests (vs. siCtrl ). E GFP fluorescence intensity distribution of Lin − Sca1 + progenitor cells 24 hours after transfection with GFP mRNA, determined by flow cytometry (compared to non‐transfected cells). F mRNA expression in female Cited4 F/F Lin − Sca1 + progenitor cells transfected with Cre or control mRNA prior to differentiation in the presence of 100 nM Rosi or vehicle for 8 days, as determined by qRT–PCR ( n = 3). t ‐test Cre vs. Ctrl (Rosi), ** P = 0.002 ( Cited4 ), * P = 0.039 ( Adipoq ), * P = 0.015 ( Ucp1 ), * P = 0.011 ( Cidea ), ** P = 0.004 ( Cpt1b ), * P = 0.011 ( Dio2 ). G mRNA expression in female Cited4 F/F Lin − Sca1 + progenitor cells transfected with Cre or control mRNA 3 days after induction of differentiation in the presence of 100 nM Rosi or vehicle for 8 days, as determined by qRT–PCR ( n = 4). t ‐test Cre vs. Ctrl, *** P = 0.0005 ( Cited4 ), ** P = 0.009 ( Cpt1b ). Data information: Data are presented as mean ± SEM except for (E) (individual data).

Article Snippet: For the transfection of primary Lin − Sca1 + cells from Cited4 F/F mice with StemMACSTM Cre recombinase (130‐101‐113; Miltenyi Biotec) or StemMACSTM Nuclear eGFP messenger RNA (mRNA) (130‐101‐119; Miltenyi Biotec), a mixture of Lipofectamine ® RNAiMAX transfection reagent (2 μl/ml) and Opti‐MEM medium was combined with Opti‐MEM containing the respective mRNA (500 ng/ml final concentration).

Techniques: Microscopy, Expressing, Quantitative RT-PCR, Transfection, Fluorescence, Flow Cytometry, Control

Journal: EMBO Molecular Medicine

Article Title: Cited4 is a sex‐biased mediator of the antidiabetic glitazone response in adipocyte progenitors

doi: 10.15252/emmm.201708613

Figure Lengend Snippet: A–C Quantitative fluorescence microscopy of LipidTOX‐ and DAPI‐stained female Cited4 F/F Lin − Sca1 + progenitor cells transfected with Cre or control mRNA prior to differentiation in the presence of 100 nM Rosi for 8 days ( n = 5). ** P = 0.002 in t ‐test (Cre vs. Ctrl). Scale bar is 10 μm. D, E Ucp1 expression in female Cited4 F/F Lin − Sca1 + progenitor cells transfected with Cre or control mRNA prior to differentiation in the presence of 100 nM Rosi for 8 days, as determined by Western blot with VCP as loading control ( n = 3). *** P = 0.0008 in t ‐test (Cre vs. Ctrl). F Cellular respiration in female Lin − Sca1 + progenitor cells differentiated in the presence of 100 nM Rosi for 8 days. The extracellular oxygen consumption rate (OCR) was determined upon injection of the indicated substances and normalized to DNA content. CL: CL‐316243. Representative experiment ( n = 9). * P = 0.041 (47 min), ** P = 0.009 (55 min), ** P = 0.003 (62 min) in 2 × 2 ANOVA with Holm–Sidak posttests ( Cited4 −/− vs. Cited4 +/+ ). G Normalized CL‐stimulated uncoupled respiration of cells in (F). Values represent the means of the three time points after CL injection after subtraction of the means of the oligomycin time points ( n = 9). * P = 0.027 in t ‐test ( Cited4 −/− vs. Cited4 +/+ ). Data information: Data are presented as mean ± SEM. Source data are available online for this figure.

Article Snippet: For the transfection of primary Lin − Sca1 + cells from Cited4 F/F mice with StemMACSTM Cre recombinase (130‐101‐113; Miltenyi Biotec) or StemMACSTM Nuclear eGFP messenger RNA (mRNA) (130‐101‐119; Miltenyi Biotec), a mixture of Lipofectamine ® RNAiMAX transfection reagent (2 μl/ml) and Opti‐MEM medium was combined with Opti‐MEM containing the respective mRNA (500 ng/ml final concentration).

Techniques: Fluorescence, Microscopy, Staining, Transfection, Control, Expressing, Western Blot, Injection

Journal: EMBO Molecular Medicine

Article Title: Cited4 is a sex‐biased mediator of the antidiabetic glitazone response in adipocyte progenitors

doi: 10.15252/emmm.201708613

Figure Lengend Snippet: A mRNA expression in wild‐type mice without treatment, as determined by qRT–PCR ( n = 6). scWAT: subcutaneous white adipose tissue; gWAT: gonadal WAT; isBAT: interscapular brown adipose tissue; SKM: gastrocnemius skeletal muscle. 2 × 2 ANOVA with Holm–Sidak posttests, *** P = 0.0004 (female vs. male), ### P = 0.0004, ### P = 7 × 10 −6 , ### P = 6 × 10 −9 , ### P = 3 × 10 −10 , (isBAT, SKM, heart, liver, respectively, vs. scWAT within indicated sex), # P = 0.032 (females: gWAT vs. scWAT), ## P = 0.006 (females: Heart vs. scWAT), ## P = 0.006 (males: Liver vs. scWAT). B Ucp1 expression in scWAT of mice fed a diet with 0.0075% Rosi or control diet for 2.5 weeks, determined by Western blot with VCP as loading control ( n = 3, same samples as in Fig C and D). C mRNA expression in BAT of mice treated as in (A), determined by qRT–PCR. t ‐test Cited4 −/− vs. Cited4 +/+ (Rosi), females: n = 5/5/6/6, males: n = 5/4/5/5, *** P = 0.0004. D mRNA expression in gWAT of mice treated as in (A), determined by qRT–PCR. t ‐test Cited4 −/− vs. Cited4 +/+ (Rosi), females: n = 5/5/6/6, males: n = 5/4/5/5. E Three‐day averages of total activity counts per day and mouse of female mice fed a diet with 0.0075% Rosi for 2.5 weeks, determined by Phenomaster ( n = 9/10, t ‐test). F Three‐day averages of food intake per day and mouse of female mice shown in (E) ( n = 9/10, t ‐test). G Oxygen consumption rate of female mice fed control diet, determined by indirect calorimetry and adjusted for body weight by ANCOVA. Three‐day averages of VO 2 were calculated for each mouse ( n = 10). ANCOVA with Bonferroni's posttest ( Cited4 −/− vs. Cited4 +/+ ). Data information: Data are presented as mean ± SEM. Source data are available online for this figure.

Article Snippet: For the transfection of primary Lin − Sca1 + cells from Cited4 F/F mice with StemMACSTM Cre recombinase (130‐101‐113; Miltenyi Biotec) or StemMACSTM Nuclear eGFP messenger RNA (mRNA) (130‐101‐119; Miltenyi Biotec), a mixture of Lipofectamine ® RNAiMAX transfection reagent (2 μl/ml) and Opti‐MEM medium was combined with Opti‐MEM containing the respective mRNA (500 ng/ml final concentration).

Techniques: Expressing, Quantitative RT-PCR, Control, Western Blot, Activity Assay

Journal: EMBO Molecular Medicine

Article Title: Cited4 is a sex‐biased mediator of the antidiabetic glitazone response in adipocyte progenitors

doi: 10.15252/emmm.201708613

Figure Lengend Snippet: A, B mRNA expression in scWAT of mice fed a diet with 0.0075% Rosi or control diet for 2.5 weeks, determined by qRT–PCR. t ‐test Cited4 −/− vs. Cited4 +/+ (Rosi), (A) n = 5/5/6/6, ** P = 0.003 ( Ucp1 ), ** P = 0.008 ( Cidea ), ** P = 0.007 ( Cyc1 ), * P = 0.048 ( Ndufb3 ), (B) n = 5/4/5/5, * P = 0.022 ( Ucp1 ), * P = 0.037 ( Cidea ). C, D Ucp1 expression in scWAT of mice treated as in (A), as determined by Western blot with VCP as loading control. t ‐test Cited4 −/− vs. Cited4 +/+ (Rosi), females: n = 5/5/6/6, * P = 0.011, males: n = 5/4/5/5. E mRNA expression in scWAT of female mice treated with CL‐316,243 (CL) (1 mg/kg/day via Alzet minipumps) or vehicle for 10 days ( n = 5/7/13, t ‐test Cited4 −/− vs. Cited4 +/+ (CL)). F mRNA expression in scWAT of female mice exposed to 5°C or 23°C for 2 weeks ( n = 5/10/6, t ‐test Cited4 −/− vs. Cited4 +/+ (5°C)). Data information: Data are presented as mean ± SEM. Source data are available online for this figure.

Article Snippet: For the transfection of primary Lin − Sca1 + cells from Cited4 F/F mice with StemMACSTM Cre recombinase (130‐101‐113; Miltenyi Biotec) or StemMACSTM Nuclear eGFP messenger RNA (mRNA) (130‐101‐119; Miltenyi Biotec), a mixture of Lipofectamine ® RNAiMAX transfection reagent (2 μl/ml) and Opti‐MEM medium was combined with Opti‐MEM containing the respective mRNA (500 ng/ml final concentration).

Techniques: Expressing, Control, Quantitative RT-PCR, Western Blot

Journal: EMBO Molecular Medicine

Article Title: Cited4 is a sex‐biased mediator of the antidiabetic glitazone response in adipocyte progenitors

doi: 10.15252/emmm.201708613

Figure Lengend Snippet: A, B mRNA expression in scWAT of mice fed a high‐fat diet (HFD) for 11 weeks followed by 5 weeks of HFD with 0.0075% Rosi (qRT–PCR). t ‐test Cited4 −/− vs. Cited4 +/+ , (A) n = 8/7, * P = 0.029 ( Ucp1 ), ** P = 0.009 ( Elovl3 ), ** P = 0.006 ( Cpt1b ), ** P = 0.004 ( Cyc1 ), * P = 0.016 ( Ndufb3 ), ** P = 0.003 ( Cd36 ), ** P = 0.009 ( Gyk ), (B) n = 7/8, * P = 0.026 ( Pck1 ). C, D Ucp1 protein expression in scWAT of mice treated as in (A), determined by Western blot with VCP as loading control ( n = 8/7 for females and n = 7/8 for males). Arrow indicates the band specific to Ucp1 (see Appendix Fig S5F ). Data information: Data presented as mean ± SEM (A and B), individual mice (D, males), or means of replicate blots with independent lysates from the same mice (D, females). Source data are available online for this figure.

Article Snippet: For the transfection of primary Lin − Sca1 + cells from Cited4 F/F mice with StemMACSTM Cre recombinase (130‐101‐113; Miltenyi Biotec) or StemMACSTM Nuclear eGFP messenger RNA (mRNA) (130‐101‐119; Miltenyi Biotec), a mixture of Lipofectamine ® RNAiMAX transfection reagent (2 μl/ml) and Opti‐MEM medium was combined with Opti‐MEM containing the respective mRNA (500 ng/ml final concentration).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Control

Journal: Nature Communications

Article Title: Naturally-occurring cholesterol analogues in lipid nanoparticles induce polymorphic shape and enhance intracellular delivery of mRNA

doi: 10.1038/s41467-020-14527-2

Figure Lengend Snippet: a Schematic showing desorption of PEG-lipids from LNPs allows ApoE binding causing LDL-mediated cellular uptake in cells following which a small portion of nucleic acids escape the endosome and the majority are recycled back by lysosomal transporters or directed to degradative endocytic compartments. b IUPAC-IUB nomenclature of (1989) cholesterol ring system with rings (A, B, C, and D), carbon numbering from C-1 to C-29, c the structure of cholesterol, and d schematic diagram showing classification of cholesterol into three regions: head, body, and tail. e Classification of cholesterol analogs- Group I-9,10-secosteroids, Group II-C-24 alkyl steroids, Group III-pentacyclic steroids. Structural variations from cholesterol are highlighted in red. f Cholesterol analogs were screened for particle size (nm), mRNA encapsulation (percent), and transfection efficiency (200 ng mRNA). Transfection experiments were conducted with n = 3. Source data are provided as a Source Data file.

Article Snippet: In vitro HeLa transfection was performed using LNPs and eLNPs containing Cyanine-5 tagged EGFP mRNA (TriLink Biotechnologies).

Techniques: Binding Assay, Encapsulation, Transfection

Journal: Nature Communications

Article Title: Naturally-occurring cholesterol analogues in lipid nanoparticles induce polymorphic shape and enhance intracellular delivery of mRNA

doi: 10.1038/s41467-020-14527-2

Figure Lengend Snippet: a – d C-24 alkyl derivatives with variations in the tail and body were screened for their effect on size, mRNA encapsulation efficiency ( EE ), and transfection efficiency of LNPs (dosed at 10–200 ng mRNA per well for C-24 alkyl derivatives in b and 50 ng mRNA per well for sequential addition of double bonds in d ). The variations of these structures from cholesterol and β-sitosterol are depicted as red and yellow, respectively. e – f Structural modification of the head group in β-sitosterol with either amino acids (polar) or acetate (non-polar) was also evaluated for its effect on nanoparticle size, encapsulation, and transfection of LNPs (200 ng mRNA per well). The color code is indicative of polarity (green), non-polarity (yellow), and structural difference from cholesterol (red). Horizontal dashed red line represents eLNP expression set to 100 percent. All experiments were conducted with n = 3; mean ± SD. Source data are provided as a Source Data file.

Article Snippet: In vitro HeLa transfection was performed using LNPs and eLNPs containing Cyanine-5 tagged EGFP mRNA (TriLink Biotechnologies).

Techniques: Encapsulation, Transfection, Modification, Expressing

Journal: Nature Communications

Article Title: Naturally-occurring cholesterol analogues in lipid nanoparticles induce polymorphic shape and enhance intracellular delivery of mRNA

doi: 10.1038/s41467-020-14527-2

Figure Lengend Snippet: a HeLa cells were exposed to β-sitosterol containing eLNPs at different doses (1–400 ng) and compared to cholesterol-containing LNPs for cellular uptake. Cy5-EGFP mRNA was used as a tracer, and high-content imaging was performed and quantified using the Cellomics software. ( n = 3; mean ± SD. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. Significance was determined using nonlinear regression with ANOVA.) Source data are provided as a Source Data file. b Uptake kinetics of LNPs and eLNPs at 10 ng mRNA per well (0–24 h) in HeLa cells. c 1st derivative of uptake of LNP and eLNP demonstrates substantially higher rate of uptake and retention in HeLa over the 24 h post-transfection. d Endosomal escape was visualized using smFISH. Representative fluorescent images showing mRNA, LNP, and image analysis after delivery with LNP or eLNP in HeLa cells. e Quantitative image analysis of the number of cytosolic mRNAs (turquoise bars) compared to the number of LNPs per cell (magenta bars) after cellular delivery. f Ratio of cytosolic mRNA delivery to total LNP uptake indicative of endosomal escape. g – j 3D-DyPLoT was performed for high spatiotemporal resolution of nanoparticle transport. g Representative trajectories of internalized LNP and eLNP. The measured 3D position is plotted at 1 ms temporal resolution. h Histograms of the log-linear efficiency for LNP (blue) and eLNP (red), both of which display a bimodal distribution of stationary and mobile. i Population of the stationary and mobile fractions for LNP and eLNP. Population was measured by fitting the histograms in h to a two-term Gaussian and extracting the area under the two modes of the distribution. n = 156 (LNP) and 286 (eLNP); mean ± SD. p = 0.0009. Significance was determined using t -test. j Best fit distributions of LNP and eLNP, showing a clear increase in the mobile fraction for eLNP.

Article Snippet: In vitro HeLa transfection was performed using LNPs and eLNPs containing Cyanine-5 tagged EGFP mRNA (TriLink Biotechnologies).

Techniques: Imaging, Software, Transfection

Journal: Nature Communications

Article Title: Naturally-occurring cholesterol analogues in lipid nanoparticles induce polymorphic shape and enhance intracellular delivery of mRNA

doi: 10.1038/s41467-020-14527-2

Figure Lengend Snippet: LNPs and eLNPs were compared for their ability to deliver mRNA to a wild-type (WT) and NPC1 knockout (NPC1 −/− ) mouse embryonic fibroblasts (200 ng mRNA per well), and b patient-derived NPC1, and c NPC2 loss-of-function fibroblasts. All experiments were conducted with n = 3; mean ± SD. (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. Significance was determined using multiple t -test.) Source data are provided as a Source Data file.

Article Snippet: In vitro HeLa transfection was performed using LNPs and eLNPs containing Cyanine-5 tagged EGFP mRNA (TriLink Biotechnologies).

Techniques: Knock-Out, Derivative Assay

Journal: Tissue Engineering. Part A

Article Title: M 3 RNA Drives Targeted Gene Delivery in Acute Myocardial Infarction

doi: 10.1089/ten.tea.2017.0445

Figure Lengend Snippet: M3RNA-mCherry expression in multiple cell lines. (A) Representative fluorescence and phase contrast images of HCF and HEK cells upon mCherry mRNA transfection at indicated time periods. Scale bar = 100 μm. (B) Fluorescence of mCherry protein expression was quantified in HDF, HCF, and HEK cells. Plots indicate mean ± SEM of average fluorescence intensity (arbitrary units) at indicated time points (n = 3). ANOVA using GraphPad Prism with Tukey's multiple comparison test revealed significant differences between 48 and 144 h in HCF and HDF (****p < 0.0001), and significant expression at all time points in all three cell lines when compared to 4 h (****p < 0.0001, **p < 0.01, and *p < 0.05). (C) Representative flow cytometry plots of HCF and HEK cells upon M3RNA-mcherry transfection. (D) Percent transfection efficiency of sorted HDF, HCF, and HEK cells was using mock-transfected cells as control and transfected cells at 4 and 24 h. Results are plotted as mean ± SEM (n = 3) of three different cell lines. ANOVA using Dunnett's multiple comparison test revealed significant differences between the 4- and 24-h transfection with M3RNA-mCherry in HCF and HEK cells (**p < 0.01, ****p < 0.0001), and significant expression levels at 4 and 24 h transfection in all three cell lines when compared to mock transfected cells (****p < 0.0001). ANOVA, analysis of variance; HCF, human cardiac fibroblasts; HDF, human dermal fibroblasts; HEK, human embryonic kidney cells; M3RNA, microencapsulated modified messenger RNA; NS, not significant.

Article Snippet: 31 , 32 In vivo delivery of FLuc, EGFP, and mCherry-modified mRNA In vivo delivery of modified mRNAs were carried out in FVB/NJ mice (18–22 g, 6–8 weeks of age) Jackson laboratory using modified protocol.

Techniques: Expressing, Fluorescence, Transfection, Comparison, Flow Cytometry, Control, Modification

Journal: Tissue Engineering. Part A

Article Title: M 3 RNA Drives Targeted Gene Delivery in Acute Myocardial Infarction

doi: 10.1089/ten.tea.2017.0445

Figure Lengend Snippet: M3RNA-mCherry expression in neonatal rat primary cardiomyocytes. (A) Representative phase contrast and fluorescence images exhibiting rapid and declining mCherry expression at indicated time points within the rat primary neonatal cardiomyocytes. Scale bar = 100 μm. (B) Quantitation of mCherry protein expression in cardiomyocytes upon transfection (n = 3 with >10 images/time point) plotted as mean ± SEM average fluorescence intensity. ANOVA using GraphPad Prism with Tukey's multiple comparison test revealed significant differences between 24, 48, and 144 h (****p < 0.0001), and significant expression levels at 24 and 48 h when compared to 4-h time point (***p < 0.001, ****p < 0.001). (C) Representative flow cytometry plots of cardiomyocytes at the indicated time points posttransfection with mCherry mRNA using mock-transfected cells as control. Transfection efficiency was calculated from sets of three different experiments (n = 3). (D) Primary cardiomyocytes were co-transfected with mCherry, GFP, and FLuc mRNA and the representative image exhibiting triple transfection in the same cells is shown. Scale bar = 100 μm. * indicates the cardiac fibroblast transfection with three genes and ** represents the nontransfected cells in the same field. FLuc, firefly luciferase; GFP, green fluorescent protein.

Article Snippet: 31 , 32 In vivo delivery of FLuc, EGFP, and mCherry-modified mRNA In vivo delivery of modified mRNAs were carried out in FVB/NJ mice (18–22 g, 6–8 weeks of age) Jackson laboratory using modified protocol.

Techniques: Expressing, Fluorescence, Quantitation Assay, Transfection, Comparison, Flow Cytometry, Control, Luciferase

Journal: Tissue Engineering. Part A

Article Title: M 3 RNA Drives Targeted Gene Delivery in Acute Myocardial Infarction

doi: 10.1089/ten.tea.2017.0445

Figure Lengend Snippet: Graphical representation of nanoparticle. Nanoparticles (∼100 nm) act as carriers of mRNA coated with positively charged biological polymers that form complexes with negatively charged mRNA molecules due to ionic interactions.

Article Snippet: 31 , 32 In vivo delivery of FLuc, EGFP, and mCherry-modified mRNA In vivo delivery of modified mRNAs were carried out in FVB/NJ mice (18–22 g, 6–8 weeks of age) Jackson laboratory using modified protocol.

Techniques:

Journal: Tissue Engineering. Part A

Article Title: M 3 RNA Drives Targeted Gene Delivery in Acute Myocardial Infarction

doi: 10.1089/ten.tea.2017.0445

Figure Lengend Snippet: Gene expression within heart upon direct myocardial injection of M3RNAs. (A) Representative bioluminescence images showing the time course of luciferase expression following direct injections of M3-FLuc mRNA into the anterior left ventricle wall. Mice were intubated and intracardiac injections were performed upon open chest surgery (see Materials and Methods). In vivo bioluminescence images of mice in the supine position are shown at 2, 24, 48, and 72 h postsurgery. (B) Quantitative bioluminescence levels as average radiance (photons/cm2) from three different experiments (five animals each; three FLuc M3RNA and two Vehicle only) were obtained from the regions of interest and were plotted against time. ****p < 0.0001, **p < 0.01. (C) IF analysis showing the mCherry expression following direct injections into the anterior left ventricle wall. mCherry-M3RNA was injected directly into the left ventricle and heart tissues were obtained 24 h postsurgery. Hearts were fixed, sectioned, and stained with anti-mCherry and troponin antibody. Upper panel shows the staining in mock-transfected hearts, while the bottom middle panel shows the mCherry expression in heart tissue (red channel), while the left panel shows the anti-mCherry antibody and the right panel is revealing the sarcomeric structures using troponin antibody. Note the structures positive for mCherry and anti mCherry, but negative for sarcomeric structures (indicated by #). (D) IF analysis of hearts (as in C above) showing GFP, mCherry, and FLuc expression upon direct injections of M3RNAs into the left ventricle and heart tissues was processed as in C above. Images were acquired using confocal microscope LSM780 with 3 × 3 tiles using the tiling tool to acquire bigger area with sharp images using a 40 × , 1.2 W lens. Upper panel shows the GFP, mCherry, and FLuc in green, red, and far-red channel, respectively, in mock-transfected hearts, while the bottom panel shows indicated gene expressions in heart tissue. In merged image, # represents areas where transfection of a single gene was observed. Scale bar = 50 μm. IF, immunofluorescence.

Article Snippet: 31 , 32 In vivo delivery of FLuc, EGFP, and mCherry-modified mRNA In vivo delivery of modified mRNAs were carried out in FVB/NJ mice (18–22 g, 6–8 weeks of age) Jackson laboratory using modified protocol.

Techniques: Gene Expression, Injection, Luciferase, Expressing, In Vivo, Staining, Transfection, Microscopy, Immunofluorescence

Journal: Tissue Engineering. Part A

Article Title: M 3 RNA Drives Targeted Gene Delivery in Acute Myocardial Infarction

doi: 10.1089/ten.tea.2017.0445

Figure Lengend Snippet: Targeted mCherry mRNA within infarcted porcine heart using alginate gel (A) Angiography of the pig heart showing the induction of acute myocardial infarction. Left panel shows the distribution of arterial blood flow along the anterior portion of the left ventricle to determine proper balloon placement. Right panel shows the inflated balloon blocking completely the distal blood flow. Scale bar = 10 mm. (B) Serial intracardiac echocardiographic images document transition of the anterior wall of the left ventricle (arrow) from normal (top) to injured after 90 min of LAD occlusion (middle) to loaded with alginate gel having M3RNA (lower panel) over 10 min immediately after reperfusion (total time 100 min). Scale bar = 10 mm. (C) Pig heart removed and flushed with chilled normal saline, and sectioned was imaged to document mCherry expression surrounding the anterior wall infarcted area in most of the areas starting from the apex to the mid-papillary portion of the left ventricle. Scale bar = 10 mm. (D) Heart areas from the anterior wall infarcted region (mCherry transfected) versus remote regions (control) demonstrate significant mCherry expression on IF probing. Total number of pig studies n = 10 for alginate optimization and n = 4 for documentation of infarct targeting. Scale bar = 100 μm. LAD, left anterior descending coronary artery; LCx, left circumflex artery.

Article Snippet: 31 , 32 In vivo delivery of FLuc, EGFP, and mCherry-modified mRNA In vivo delivery of modified mRNAs were carried out in FVB/NJ mice (18–22 g, 6–8 weeks of age) Jackson laboratory using modified protocol.

Techniques: Blocking Assay, Saline, Expressing, Transfection, Control

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Serum factors create species-specific barriers to hepatic gene transfer by lipid nanoparticles in liver-humanized mice

doi: 10.1016/j.omtm.2025.101470

Figure Lengend Snippet: Experimental setup and DasherGFP expression in chimeric livers Study timelines (A) and relative human hepatocyte engraftment levels (B) for individual liver-humanized NSG-PiZ and FRG mice. Relative levels of human hepatocyte engraftment were determined prior to administration of PBS or LNPs (NSG-PiZ - 2 weeks pre-administration; FRG - 1 week pre-administration) via analysis of human albumin (HuAlb) levels in serum. Livers from individual NSG-PiZ (C and E) and FRG (D and F) mice at 24 h post treatment with PBS or LNP1, LNP2, or LNP3 formulated with DasherGFP mRNA were stained for GFP expression via immunofluorescence. DasherGFP expression was detected in paraffin sections with an anti-CometGFP antibody. For NSG-PiZ mice, parts of the left lateral, left medial, and right medial lobes are shown. For FRG mice, part of the left lateral lobe is shown. Scale bars, 5 mm. For NSG-PiZ (C) and FRG (D) mice, DasherGFP fluorescence was quantified across all DAPI+ cells, and DAPI+/DasherGFP+ cells were classified as low, medium, and high expressors. Individual mouse IDs are indicated in the top left (C and D). Error bars (E and F) indicate standard deviations.

Article Snippet: EGFP mRNA (m1Ψ)—SM-102 LNP, EGFP mRNA (m1Ψ)—ALC0315 LNP and EGFP mRNA (m1Ψ)—LP01 LNP were purchased from Genscript (Cat# SC2346).

Techniques: Expressing, Staining, Immunofluorescence, Fluorescence

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Serum factors create species-specific barriers to hepatic gene transfer by lipid nanoparticles in liver-humanized mice

doi: 10.1016/j.omtm.2025.101470

Figure Lengend Snippet: LNPs efficiently transfect mouse but not human hepatocytes in liver-humanized NSG-PiZ and FRG mice Livers from a single chimeric NSG-PiZ (A, mouse 4-3) or FRG (B, mouse #6) mouse at 24 h post treatment with PBS or LNP formulated with DasherGFP mRNA were stained with H&E (left images) or by immunofluorescence in paraffin sections with antibodies that detect DasherGFP, hCK18, or FAH. For mouse 4-3, serial sections were stained with H&E or co-labeled with DasherGFP and hCK18. For mouse #6, serial sections were stained with H&E, FAH, and DasherGFP, respectively. DasherGFP expression was detected with an anti-CometGFP antibody. Scale bars, upper images, 5 mm; middle images, 500 μm; lower images, 100 μm.

Article Snippet: EGFP mRNA (m1Ψ)—SM-102 LNP, EGFP mRNA (m1Ψ)—ALC0315 LNP and EGFP mRNA (m1Ψ)—LP01 LNP were purchased from Genscript (Cat# SC2346).

Techniques: Staining, Immunofluorescence, Labeling, Expressing

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Serum factors create species-specific barriers to hepatic gene transfer by lipid nanoparticles in liver-humanized mice

doi: 10.1016/j.omtm.2025.101470

Figure Lengend Snippet: Biodistribution of DasherGFP mRNA in liver-humanized NSG-PiZ and FRG mouse liver RNAscope was performed on sections of livers from chimeric NSG-PiZ (A) or FRG (B and C) mice at 24 h post treatment with PBS or LNP1, LNP2, or LNP3 formulated with DasherGFP mRNA. High-power images of mouse hepatocytes from LNP2-treated (white arrows) but not LNP3-treated (red arrows) FRG mice demonstrate diffuse intracellular DasherGFP mRNA signal (C). Individual mouse IDs are indicated on the left. Sections were hybridized with probes specific for DasherGFP (green), human B2M (purple), and mouse GAPDH (cyan) and stained with DAPI (blue, A and B; white, C). Scale bars, 50 μm.

Article Snippet: EGFP mRNA (m1Ψ)—SM-102 LNP, EGFP mRNA (m1Ψ)—ALC0315 LNP and EGFP mRNA (m1Ψ)—LP01 LNP were purchased from Genscript (Cat# SC2346).

Techniques: RNAscope, Staining

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Serum factors create species-specific barriers to hepatic gene transfer by lipid nanoparticles in liver-humanized mice

doi: 10.1016/j.omtm.2025.101470

Figure Lengend Snippet: Comparative histopathology and putative LNP receptor expression in chimeric NSG-PiZ and FRG mouse livers Livers from liver-humanized NSG-PiZ or FRG mice at 24- or 48-h post treatment with PBS, or LNP1–3 formulated with DasherGFP mRNA (A and B). Paraffin-embedded sections from representative livers were stained with H&E (A). Scale bar, 100 μm. Frozen sections from representative livers were stained with Oil red O and counterstained with hematoxylin (B). Scale bars, 1 mm (inset image) and 100 μm (main image). Dashed lines indicate approximate borders between human and mouse hepatocytes. H, human hepatocytes; M, mouse hepatocytes. snRNA-seq and scRNA-seq analysis of putative LNP receptor mRNA levels in hepatocytes of chimeric FRG and NSG-PiZ mice (C). (D) Detection of ALB , A PO E , LDLR , ASGR1 , and ASGR2 mRNA in human and mouse hepatocytes from NSG-PiZ and FRG mice. (E) The percentage of total reads per cell for ALB , APOE , LDLR , ASGR1 , and ASGR2 in human (H) or mouse (M) hepatocytes determined by scRNA-seq (NSG-PiZ) or snRNA-seq (FRG). For the scRNA-seq analysis, cells were pooled from four low engrafted chimeric NSG-PiZ mice prior to single-cell analysis. For the snRNA-seq analysis, reads were pooled from two highly engrafted chimeric FRG mice or two low engrafted chimeric FRG mice from a previously published dataset (Bioproject: PRJNA857105; GEO: GSM6319631 , GSM6319632 , GSM6319637 , GSM6319638 ; Cabanes-Creus et al. ). FRG reads were processed as previously described (Cabanes-Creus et al. ) and scRNA-seq reads were processed as described in the methods. (C) was created using Biorender.

Article Snippet: EGFP mRNA (m1Ψ)—SM-102 LNP, EGFP mRNA (m1Ψ)—ALC0315 LNP and EGFP mRNA (m1Ψ)—LP01 LNP were purchased from Genscript (Cat# SC2346).

Techniques: Histopathology, Expressing, Staining, Single-cell Analysis

Journal: PLoS Biology

Article Title: Identification of 3′ UTR motifs required for mRNA localization to myelin sheaths in vivo

doi: 10.1371/journal.pbio.3001053

Figure Lengend Snippet: (A) Schematic of the MS2 system to visualize mRNA localization in oligodendrocytes. sox10 regulatory DNA drives expression of nuclear-localized MS2 coat protein, NLS-MCP-EGFP (orange crescent and green star). mbpa regulatory elements drive expression of mRNA encoding mScarlet-CAAX fluorescent protein with a repetitive sequence that creates 24 stem loops (24xMBS). When co-expressed, the mRNA–protein complex is exported from the nucleus and localized via the 3′ UTR. (B) Schematic of MS2 expression plasmids used for transient expression in oligodendrocytes with target sequences for Tol2 transposase to facilitate transgene integration. (C and D) Representative images of localization directed by the mbpa (C) or control sv40 3′ UTR (D). Asterisks mark cell bodies with high expression levels of the nuclear-localized MCP-EGFP. Boxed areas are enlarged to highlight sheath termini (arrows). (E) Average mRNA abundance per myelin sheath, measured by EGFP fluorescence intensity normalized to the average intensity of the sv40 control. sv40 : n = 5 larvae, 35 sheaths. mbpa : n = 6 larvae, 38 sheaths. (F) Average mRNA abundance per soma, measured by EGFP fluorescence intensity normalized to the average intensity of the sv40 control. sv40 : n = 11 larvae, 20 cell bodies. mbpa : n = 15 larvae, 21 cell bodies. (G and H) Representative images of 2 myelinating oligodendrocytes expressing mRNA lacking the 24xMBS . NLS-MCP-EGFP remains in the nucleus at 3 dpf (G) and 5 dpf (H). Scale bars, 10 μm. Statistical significance evaluated using Wilcoxon test. The underlying numerical data can be found in and Data. 3' UTR, 3' untranslated region; dpf, days post fertilization.

Article Snippet: The EGFP smFISH probes were purchased from Stellaris LGC Biosearch Technologies.

Techniques: Expressing, Sequencing, Control, Fluorescence

Journal: PLoS Biology

Article Title: Identification of 3′ UTR motifs required for mRNA localization to myelin sheaths in vivo

doi: 10.1371/journal.pbio.3001053

Figure Lengend Snippet: (A and B) Representative images of smFISH experiments using 4 dpf transgenic larva expressing EGFP-CAAX to mark oligodendrocytes. Images show sagittal sections of the hindbrain. DAPI stain labels nuclei. Sections were treated with smFISH probes designed to detect mbpa (A) or egfp (B) mRNA. Asterisks mark cell bodies and brackets mark myelin tracts. Scale bars, 10 μm. (C) Average mbpa mRNA density per cell body or equivalent volume of myelin from 3 to 5 dpf. Density was measured using the integrated density of fluorescence intensity in cell bodies and approximately equal volumes of myelin along the myelin tracts. A minimum (n) for each group was 3 larvae, 6 cell bodies, and 15 myelin regions. Statistical significance evaluated using Wilcoxon test. (D) Proportion of egfp or mbpa mRNA abundance in cell bodies compared to myelin tracts. A minimum (n) for each group was 3 larvae, 11 cell bodies, and 21 myelin regions. (E) Average mbpa mRNA density within individual sheaths plotted as a function of sheath length. Statistical significance evaluated using Spearman’s correlation coefficient. Shaded area represents 95% confidence interval. n = 7 embryos, 26 sheaths. The underlying numerical data can be found in – Data. dpf, days post fertilization; smFISH, single molecule fluorescent in situ hybridization.

Article Snippet: The EGFP smFISH probes were purchased from Stellaris LGC Biosearch Technologies.

Techniques: Transgenic Assay, Expressing, Staining, Fluorescence, In Situ Hybridization

Journal: PLoS Biology

Article Title: Identification of 3′ UTR motifs required for mRNA localization to myelin sheaths in vivo

doi: 10.1371/journal.pbio.3001053

Figure Lengend Snippet: (A) smFISH images of a single optical section of a myelin sheath in a 3-dpf larva spinal cord. mbpa transcripts line the myelin sheath. Arrows highlight clusters of mbpa mRNA transcripts. (B) smFISH images of a single optical section of myelin tracts in the hindbrain of a 5-dpf larva. Boxed area magnified to highlight sheath termini (arrows). (C) smFISH images of a single optical section in transverse plane of myelin sheaths in a 5-dpf larva midbrain. Scale bars (A, B, and D), 5 μm; (C, boxed enlargements), 1 μm. (D) Representative images from MS2 system showing colocalization of mRNA containing mbpa 3′ UTR and F-actin in a myelinating oligodendrocyte. Asterisk marks the cell body, and boxes are magnified to highlight sheath termini. Arrows highlight sheaths with mRNA, and arrowheads highlight sheaths lacking mRNA. (E) Proportion of sheaths with mRNA enriched in sheath termini at 4 dpf using the MS2 system. Proportion measured as (sheaths with enrichment / number of sheaths) = 10/35 sv40 , 18/38 mbpa . (F) Average fluorescence intensity of MS2 mRNA reporter containing the sv40 or mbpa 3′ UTRs across a 7-μm distance, at 0.2-μm intervals, from myelin sheath termini at 4 dpf. Each line scan was normalized to the average fluorescent intensity per sheath. All normalized values for each distance were then averaged. Shaded area represents 95% confidence interval. Statistical significance was evaluated every 0.2 μm using Wilcoxon test, and the distance between 0.8–1.0 μm was statistically significant (blue line). sv40 3′ UTR n = 5 larvae, 35 sheaths. mbpa 3′ UTR n = 6 larvae, 38 sheaths. The underlying numerical data can be found in and Data. 3' UTR, 3' untranslated region; dpf, days post fertilization; F-actin, filamentous actin; smFISH, single molecule fluorescent in situ hybridization.

Article Snippet: The EGFP smFISH probes were purchased from Stellaris LGC Biosearch Technologies.

Techniques: Fluorescence, In Situ Hybridization

Journal: PLoS Biology

Article Title: Identification of 3′ UTR motifs required for mRNA localization to myelin sheaths in vivo

doi: 10.1371/journal.pbio.3001053

Figure Lengend Snippet: (A) Work flow to identify 3′ UTR candidates from RNA-seq data [ , , ]. (B) Representative images from MS2 system showing localization of mRNAs containing different 3′ UTR sequences in oligodendrocytes. Asterisks mark cell bodies. Scale bars, 10 μm. (C) Table listing candidate 3′ UTRs incorporated into the MS2 system, 3′ UTR length, and the percentage of sequence that was cloned based on the annotated genome (GRCz11). (D) Average mRNA abundance, measured by average EGFP fluorescent intensity, per myelin sheath for each 3′ UTR. Normalized to sv40 control, statistical significance evaluated using Wilcoxon test. A minimum (n) of 5 larvae and 18 sheaths were used in each condition at 4 dpf. The underlying numerical data can be found in and Data. 3' UTR, 3' untranslated region; dpf, days post fertilization; RNA-seq, RNA sequencing.

Article Snippet: The EGFP smFISH probes were purchased from Stellaris LGC Biosearch Technologies.

Techniques: RNA Sequencing, Sequencing, Clone Assay, Control

Journal: PLoS Biology

Article Title: Identification of 3′ UTR motifs required for mRNA localization to myelin sheaths in vivo

doi: 10.1371/journal.pbio.3001053

Figure Lengend Snippet: Representative images of smFISH experiments to visualize egfp , eif4ebp2 , or fmr1 mRNA localization at 4 dpf (A and B) and 5 dpf (C and D) in sagittal sections of hindbrain (A, C) or transverse sections of the Mauthner axon in the spinal cord (B, D). Dashed lines outline cell bodies marked by EGFP-CAAX. Scale bars, 5 μm (A, C) or 1 μm (B, D). dpf, days post fertilization; smFISH, single molecule fluorescent in situ hybridization.

Article Snippet: The EGFP smFISH probes were purchased from Stellaris LGC Biosearch Technologies.

Techniques: In Situ Hybridization

Journal: bioRxiv

Article Title: High throughput intracellular delivery by viscoelastic mechanoporation

doi: 10.1101/2023.04.24.538131

Figure Lengend Snippet: (a) eGFP expression 24 hours after mRNA transfection to Jurkat cells for a range of extracellular mRNA concentrations. (b) T cell receptor (TCR) expression in Jurkat cells 2 days after transfection of Cas9 RNP complexes designed to knock out the TCR, for a range of RNP concentrations ( n = 3 for experimental conditions, n = 1 for controls). (c) Viability and delivery of 70 kDa FITC-dextran to HEK293T cells, 24 hours after processing, for three delivery buffer compositions and 2 different chip pressures (Unstretched, n = 2 ; PBS, n = 1 ; Cytomix buffers, n = 3 ). (d) Viability and delivery of 70 kDa FITC-dextran to primary activated T cells, about 90 minutes after processing, for increasing chip pressures ( n = 2 ).

Article Snippet: For mRNA transfection, we used an mRNA encoding enhanced green fluorescent protein (eGFP) with ARCA cap modifications (Apexbio Technologies, Fisher Scientific #50-199-8310).

Techniques: Expressing, Transfection, Knock-Out