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: Human: MRC5 , ATCC , CCL-171.

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: Human: MRC5 , ATCC , CCL-171.

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: Human: MRC5 , ATCC , CCL-171.

Techniques: Knockdown, Knock-Out, Transfection, Positive Control, Control, Western Blot, Over Expression, Glo Assay, Viability Assay, 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: Intracellular accumulation of CCN1 induces autophagy through ROS production (A) MRC5 WT cells are treated with either 15 μg/mL BFA (bottom) or left untreated (top), followed by immunofluorescent staining for CCN1. The stained samples are imaged using confocal microscopy, with CCN1 fluorescence shown in green and nuclei labeled in blue using DAPI. Images are analyzed using ImageJ software. Scale bars, 10 μm. (B) Western blot analysis of intracellular CCN1 and LC3B-II protein levels in DDI2 KO and control cells treated with 15 μg/mL BFA, 60 μM CQ, or a combination of both for 24 h. (C) Measurement of autophagic flux in CCN1 KO and control cells following treatment with 15 μg/mL BFA, 60 μM CQ, or both for 24 h. (D) Western blot analysis of conditioned media to evaluate secreted CCN1, and validate the generation of MRC5 CCN1 ΔSP and WT CCN1-overexpressing cells. β-Actin from corresponding cell lysates is used as a loading control, reflecting equal numbers of plated cells. (E) Measurement of autophagic flux in MRC5 CCN1 ΔSP and WT CCN1 cells compared to control cells following treatment with 60 μM CQ for 24 h. (F) Western blot analysis of LC3B-II protein levels in CCN1 OE and control cells treated with 15 μg/mL BFA, 0.5 mM NAC, or a combination of both for 24 h. β-Actin or GAPDH is used as a loading control for all Western blots, and corresponding densitometric bar graphs are shown. Quantification of LC3B-II and CCN1 protein levels is normalized to the respective loading controls. 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: Human: MRC5 , ATCC , CCL-171.

Techniques: Staining, Confocal Microscopy, Fluorescence, Labeling, Software, 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 interacts with CCN1 and p97 (A) MRC5 and MIA PaCa-2 cells, either DDI2 KO or control, are treated with DMSO or CB-5083 (10 μM for 3 h). CCN1 protein levels are analyzed by immunoblotting using antibodies specific to DDI2, CCN1, and β-Actin, and representative blots with corresponding densitometric bar graphs are shown. (B) HEK293 cells transiently expressing protease-dead DDI2 (Flag-dRVP DDI2) are subjected to immunoprecipitation using anti-FLAG beads, followed by immunoblotting with antibodies specific to DDI2, CCN1, p97, and RAD23A. Lysate lanes are loaded with 5% of the total input used for immunoprecipitation. The molecular weights of the proteins analyzed are as follows: P97/VCP (89 kDa), RAD23A (52 kDa). (C) Representative confocal immunofluorescence microscopy images show the co-localization of DDI2 (red) and CCN1 (green) in wild-type MRC5 and MIA PaCa-2 cells. The nuclei are visualized by staining with DAPI (blue). Scale bars, 10 μm. 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: Human: MRC5 , ATCC , CCL-171.

Techniques: Control, Western Blot, Expressing, Immunoprecipitation, Immunofluorescence, Microscopy, Staining, 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: CCN1 associates with the lysosome, and DDI2 directs CCN1 toward lysosomal degradation (A) Co-immunoprecipitation of CCN1 and LAMP1 is performed in HEK293 cells without or (B) with CCN1 overexpression, using protein A/G agarose beads. Western blot analysis is applied with specific antibodies for CCN1 and LAMP1 under high- and low-contrast imaging conditions. The molecular weight of LAMP1 is 90–120 kDa (glycosylated). (C) Immunofluorescence and confocal microscopy are used to assess CCN1 colocalization with LAMP1 in MIA PaCa-2 and MRC5 wild-type cells. CCN1 is stained green in MIA PaCa-2 cells and red in MRC5 cells, while LAMP1 is stained red in MIA PaCa-2 cells and green in MRC5 cells, with nuclei visualized by DAPI staining (blue). Scale bars, 10 μm. (D) Validation of CCN1-V5 tag overexpression in MRC5 DDI2 KO cells compared to the control by Western blotting. (E) Confocal immunofluorescence microscopy images of MRC5 DDI2 KO cells overexpressing CCN1-V5 tag, and MRC5 WT cells overexpressing CCN1-V5 tag. Fixed cells are probed for CCN1-V5 tag (red) and LAMP1 (green) and stained with DAPI (blue) to visualize nuclei. Scale bars, 10 μm. Corresponding immunoblots, including molecular weight marker lanes, are provided in .

Article Snippet: Human: MRC5 , ATCC , CCL-171.

Techniques: Immunoprecipitation, Over Expression, Western Blot, Imaging, Molecular Weight, Immunofluorescence, Confocal Microscopy, Staining, Biomarker Discovery, Control, Microscopy, 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 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: NIH-3T3 (male mouse, CRL-1658; ATCC), MRC5 (male human, CCL-171; ATCC), MIA PaCa-2 (male human, CRL-1420; ATCC), HEK293T (female human, CRL-3216; ATCC) cell lines, along with their derivatives, were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals), 1X penicillin-streptomycin (Pen/Strep; Invitrogen), and 5 μg/mL Plasmocin Prophylactic (PP; InvivoGen).

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: NIH-3T3 (male mouse, CRL-1658; ATCC), MRC5 (male human, CCL-171; ATCC), MIA PaCa-2 (male human, CRL-1420; ATCC), HEK293T (female human, CRL-3216; ATCC) cell lines, along with their derivatives, were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals), 1X penicillin-streptomycin (Pen/Strep; Invitrogen), and 5 μg/mL Plasmocin Prophylactic (PP; InvivoGen).

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: NIH-3T3 (male mouse, CRL-1658; ATCC), MRC5 (male human, CCL-171; ATCC), MIA PaCa-2 (male human, CRL-1420; ATCC), HEK293T (female human, CRL-3216; ATCC) cell lines, along with their derivatives, were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals), 1X penicillin-streptomycin (Pen/Strep; Invitrogen), and 5 μg/mL Plasmocin Prophylactic (PP; InvivoGen).

Techniques: Knockdown, Knock-Out, Transfection, Positive Control, Control, Western Blot, Over Expression, Glo Assay, Viability Assay, 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: Intracellular accumulation of CCN1 induces autophagy through ROS production (A) MRC5 WT cells are treated with either 15 μg/mL BFA (bottom) or left untreated (top), followed by immunofluorescent staining for CCN1. The stained samples are imaged using confocal microscopy, with CCN1 fluorescence shown in green and nuclei labeled in blue using DAPI. Images are analyzed using ImageJ software. Scale bars, 10 μm. (B) Western blot analysis of intracellular CCN1 and LC3B-II protein levels in DDI2 KO and control cells treated with 15 μg/mL BFA, 60 μM CQ, or a combination of both for 24 h. (C) Measurement of autophagic flux in CCN1 KO and control cells following treatment with 15 μg/mL BFA, 60 μM CQ, or both for 24 h. (D) Western blot analysis of conditioned media to evaluate secreted CCN1, and validate the generation of MRC5 CCN1 ΔSP and WT CCN1-overexpressing cells. β-Actin from corresponding cell lysates is used as a loading control, reflecting equal numbers of plated cells. (E) Measurement of autophagic flux in MRC5 CCN1 ΔSP and WT CCN1 cells compared to control cells following treatment with 60 μM CQ for 24 h. (F) Western blot analysis of LC3B-II protein levels in CCN1 OE and control cells treated with 15 μg/mL BFA, 0.5 mM NAC, or a combination of both for 24 h. β-Actin or GAPDH is used as a loading control for all Western blots, and corresponding densitometric bar graphs are shown. Quantification of LC3B-II and CCN1 protein levels is normalized to the respective loading controls. 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: NIH-3T3 (male mouse, CRL-1658; ATCC), MRC5 (male human, CCL-171; ATCC), MIA PaCa-2 (male human, CRL-1420; ATCC), HEK293T (female human, CRL-3216; ATCC) cell lines, along with their derivatives, were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals), 1X penicillin-streptomycin (Pen/Strep; Invitrogen), and 5 μg/mL Plasmocin Prophylactic (PP; InvivoGen).

Techniques: Staining, Confocal Microscopy, Fluorescence, Labeling, Software, 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 interacts with CCN1 and p97 (A) MRC5 and MIA PaCa-2 cells, either DDI2 KO or control, are treated with DMSO or CB-5083 (10 μM for 3 h). CCN1 protein levels are analyzed by immunoblotting using antibodies specific to DDI2, CCN1, and β-Actin, and representative blots with corresponding densitometric bar graphs are shown. (B) HEK293 cells transiently expressing protease-dead DDI2 (Flag-dRVP DDI2) are subjected to immunoprecipitation using anti-FLAG beads, followed by immunoblotting with antibodies specific to DDI2, CCN1, p97, and RAD23A. Lysate lanes are loaded with 5% of the total input used for immunoprecipitation. The molecular weights of the proteins analyzed are as follows: P97/VCP (89 kDa), RAD23A (52 kDa). (C) Representative confocal immunofluorescence microscopy images show the co-localization of DDI2 (red) and CCN1 (green) in wild-type MRC5 and MIA PaCa-2 cells. The nuclei are visualized by staining with DAPI (blue). Scale bars, 10 μm. 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: NIH-3T3 (male mouse, CRL-1658; ATCC), MRC5 (male human, CCL-171; ATCC), MIA PaCa-2 (male human, CRL-1420; ATCC), HEK293T (female human, CRL-3216; ATCC) cell lines, along with their derivatives, were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals), 1X penicillin-streptomycin (Pen/Strep; Invitrogen), and 5 μg/mL Plasmocin Prophylactic (PP; InvivoGen).

Techniques: Control, Western Blot, Expressing, Immunoprecipitation, Immunofluorescence, Microscopy, Staining, 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: CCN1 associates with the lysosome, and DDI2 directs CCN1 toward lysosomal degradation (A) Co-immunoprecipitation of CCN1 and LAMP1 is performed in HEK293 cells without or (B) with CCN1 overexpression, using protein A/G agarose beads. Western blot analysis is applied with specific antibodies for CCN1 and LAMP1 under high- and low-contrast imaging conditions. The molecular weight of LAMP1 is 90–120 kDa (glycosylated). (C) Immunofluorescence and confocal microscopy are used to assess CCN1 colocalization with LAMP1 in MIA PaCa-2 and MRC5 wild-type cells. CCN1 is stained green in MIA PaCa-2 cells and red in MRC5 cells, while LAMP1 is stained red in MIA PaCa-2 cells and green in MRC5 cells, with nuclei visualized by DAPI staining (blue). Scale bars, 10 μm. (D) Validation of CCN1-V5 tag overexpression in MRC5 DDI2 KO cells compared to the control by Western blotting. (E) Confocal immunofluorescence microscopy images of MRC5 DDI2 KO cells overexpressing CCN1-V5 tag, and MRC5 WT cells overexpressing CCN1-V5 tag. Fixed cells are probed for CCN1-V5 tag (red) and LAMP1 (green) and stained with DAPI (blue) to visualize nuclei. Scale bars, 10 μm. Corresponding immunoblots, including molecular weight marker lanes, are provided in .

Article Snippet: NIH-3T3 (male mouse, CRL-1658; ATCC), MRC5 (male human, CCL-171; ATCC), MIA PaCa-2 (male human, CRL-1420; ATCC), HEK293T (female human, CRL-3216; ATCC) cell lines, along with their derivatives, were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals), 1X penicillin-streptomycin (Pen/Strep; Invitrogen), and 5 μg/mL Plasmocin Prophylactic (PP; InvivoGen).

Techniques: Immunoprecipitation, Over Expression, Western Blot, Imaging, Molecular Weight, Immunofluorescence, Confocal Microscopy, Staining, Biomarker Discovery, Control, Microscopy, Marker