mouse embryonic fibroblast cell line balb 3t3  (ATCC)

Journal: iScience

Article Title: Loss of DDI2 rewires proteostasis through CCN1-driven compensatory autophagy

doi: 10.1016/j.isci.2026.115056

Figure Lengend Snippet: DDI2 depletion leads to autophagy induction (A) Western blot analysis confirms the generation of NIH-3T3 DDI2 KO cells. (B) TEM images of NIH-3T3 control and DDI2 KO cells. N: nucleus. Yellow arrows: early autolysosomes. Blue arrows: late autolysosomes. Scale bars, 2 μm. (C) Autophagy flux analysis in NIH-3T3, MRC5, HAP1, ES1, EW16, and MIA PaCa-2 cells, either control or DDI2-deficient. Cells are treated with CQ (60 μM) and evaluated for LC3B-II protein levels through immunoblotting. β-Actin or GAPDH is used as the loading control. Quantification of LC3B-II protein levels is normalized to the respective loading controls, and corresponding densitometric bar graphs are shown. The molecular weights of the proteins analyzed are as follows: DDI2 (∼45 kDa), LC3B-II (∼16 kDa), β-actin (∼45 kDa), and GAPDH (∼36 kDa). Three biological replicates for each cell line are used to perform Western blotting. Statistical significance of each condition compared to the indicated control or treatment is determined using unpaired Student’s t test or two-way ANOVA with Šídák’s post hoc test, as appropriate. Data are represented as mean ± SEM. Significance levels are indicated as follows: ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. Corresponding immunoblots, including molecular weight marker lanes, are provided in .

Article Snippet: Mouse: NIH-3T3 , ATCC , CRL-1658.

Techniques: Western Blot, Control, Molecular Weight, Marker

Journal: iScience

Article Title: Loss of DDI2 rewires proteostasis through CCN1-driven compensatory autophagy

doi: 10.1016/j.isci.2026.115056

Figure Lengend Snippet: DDI2 deficiency increases CCN1 protein levels without affecting CCN1 transcription (A) Volcano plots show protein abundances in HAP1 cells in the absence of DDI2. p -values are calculated using a two-tailed unpaired t test with unequal variance. The negative log10 of the p -values is plotted on the Y axis, and the log2 fold changes are plotted on the X axis. The plots are generated using VolcaNoseR. (B) Western blot analysis of CCN1 protein levels in NIH-3T3, MRC5, ES1, EW16 and MIA PaCa-2 control, or DDI2-deficient cells treated with or without 60 μM CQ for 24 h. β-Actin or GAPDH is used as a loading control. Quantification of CCN1 protein levels is normalized to the respective loading controls, and corresponding densitometric bar graphs are shown. The molecular weight of CCN1 is 41 kDa. (C) CCN1 expression in MRC5 control and DDI2 KO cells is assessed by immunofluorescence staining. Confocal microscopy is applied to visualize CCN1 localization (green), and CCN1 fluorescence intensity is quantified using ImageJ software. Nuclei are shown in blue through staining with DAPI. Scale bars, 10 μm. (D) qRT-PCR analysis is performed to assess CCN1 mRNA levels in NIH-3T3, MRC5, and MIA PaCa-2 DDI2 KO and control cells, following treatment with either vehicle or 60 μM CQ for 24 h. Gene-specific primers are used as described in the , with 18S rRNA or GAPDH for normalization. Three biological replicates for each cell line are used to perform qRT-PCR and Western blotting. Statistical significance of each condition compared to the indicated control or treatment is determined using unpaired Student’s t test or two-way ANOVA with Šídák’s post hoc test, as appropriate. Data are represented as mean ± SEM. Significance levels are indicated as follows: ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. Corresponding immunoblots, including molecular weight marker lanes, are provided in .

Article Snippet: Mouse: NIH-3T3 , ATCC , CRL-1658.

Techniques: Two Tailed Test, Generated, Western Blot, Control, Molecular Weight, Expressing, Immunofluorescence, Staining, Confocal Microscopy, Fluorescence, Software, Quantitative RT-PCR, Marker

Journal: iScience

Article Title: Loss of DDI2 rewires proteostasis through CCN1-driven compensatory autophagy

doi: 10.1016/j.isci.2026.115056

Figure Lengend Snippet: CCN1 is required and sufficient to induce autophagy (A) Autophagy flux analysis following CCN1 knockdown via siRNA in NIH-3T3 DDI2 KO cells and CCN1 knockout using viral particles in MIA PaCa-2 DDI2 KO cells, with the subsequent treatment of the cells with CQ (60 μM) for 24 h. For siRNA transfection, GAPDH is used as a positive control. (B) NIH-3T3, MRC5, and MIA PaCa-2 cells, either control or overexpressing CCN1, are treated with CQ (60 μM) for 24 h, and autophagy flux is measured by Western blot. (C) Autophagy flux analysis in MIA PaCa-2 DDI2 KO cells following CCN1 overexpression, treated with or without CQ (60 μM) for 24 h. (D) MIA PaCa-2 controls, DDI2 KO , and DDI2 KO cells overexpressing CCN1 are treated with or without 50 nM CFZ for 16 h, and cell lysates are analyzed by Western blot using the indicated antibodies. (E) MIA PaCa-2 controls, DDI2 KO , and DDI2 KO cells overexpressing CCN1 are assessed for cell viability using the luminescent CellTiter-Glo assay. β-actin or GAPDH is used as a loading control for Western blotting, and corresponding densitometric bar graphs are shown. Quantification of LC3B-II protein levels is normalized to the respective loading controls. Each experiment is performed in three biological replicates for Western blotting and six replicates for the cell viability assay. Statistical significance of each condition compared to the indicated control or treatment is determined using unpaired Student’s t test or two-way ANOVA with Tukey’s or Šídák’s post hoc test, as appropriate. Data are represented as mean ± SEM. Significance levels are indicated as follows: ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001; ns, not significant. Corresponding immunoblots including molecular weight marker lanes, are provided in .

Article Snippet: Mouse: NIH-3T3 , ATCC , CRL-1658.

Techniques: Knockdown, Knock-Out, Transfection, Positive Control, Control, Western Blot, Over Expression, Glo Assay, Viability Assay, Molecular Weight, Marker

Journal: Materials Today Bio

Article Title: Feasibility of combining JAK1 gene editing via CRISPR-CasRx with EGCG–lactoferrin nanoparticle therapy in a microneedle-based platform for atopic dermatitis

doi: 10.1016/j.mtbio.2026.102884

Figure Lengend Snippet: Characterization and gene editing efficiency of PBAE-Plasmid NPs. ( a ) and ( b ) RNA silencing effects of different sgRNAs targeting JAK1 in NIH-3T3 and DC 2.4 cells. ( c ) Size and zeta potential of the PBAE-plasmid complex at various mass ratios. ( d ) Agarose gel electrophoresis of the PBAE/plasmid complex at different mass ratios. ( e ) Size distribution analyzed by dynamic light scattering (DLS) and transmission electron microscopy (TEM) images of PBAE-plasmid NPs at a mass ratio of 20:1. ( f ) and ( g ) Effects of the PBAE-plasmid complex at various mass ratios on NIH-3T3 and DC 2.4 cell viability. ( h ) Green fluorescence in NIH-3T3 cells transfected with PBAE-plasmid NPs. ( i ) JAK1 mRNA expression in NIH-3T3 cells transfected with PBAE-plasmid NPs. ( j ) JAK1 protein expression in mice transfected with PBAE-plasmid NPs. ( k ) Quantitative analysis of (j). Data are presented as mean ± SD (n = 3). Bars sharing the same letter are not significantly different, whereas those with different letters are statistically significant (p < 0.05).

Article Snippet: Mouse embryonic fibroblast NIH/3T3 cells and mouse dendritic DC2.4 cells were obtained from the American Type Culture Collection (ATCC).

Techniques: Plasmid Preparation, Zeta Potential Analyzer, Agarose Gel Electrophoresis, Transmission Assay, Electron Microscopy, Fluorescence, Transfection, Expressing