Journal: Cancer letters

Article Title: REDD1 is a determinant of the sensitivity of renal cell carcinoma cells to autophagy inhibition that can be therapeutically exploited by targeting PIM activity.

doi: 10.1016/j.canlet.2025.217496

Figure Lengend Snippet: Fig. 1. TP-3654 has selective anti-RCC effects and potently induces REDD1 expression. (A) TP-3654 selectively antagonizes RCC cell viability. Normal RPTEC renal cells and a panel of 6 RCC cell lines (Achn, A498, 786-O, RCC4, Caki-1 and Caki-2) were treated with the indicated concentrations of TP-3654 for 72 h. Cell viability was determined by MTT assay. Mean ± SD, n = 3. (B) TP-3654 diminishes ATP levels in RCC cells. 786-O, A498, Achn, and Caki-2 cells were treated with the indicated concentrations of TP-3654 for 72 h. Cellular ATP levels were quantified using the ATPLite assay according to the manufacturer’s directions. Mean ± SD, n = 3. (C) Effects of TP-3654 treatment on the transcriptome of RCC cells. 786-O cells were treated with 3 μM TP-3654 for 24 h. Cells were subjected to RNASeq analyses. Heatmap depicts the most significantly induced genes following TP-3654 treatment. DDIT4 (REDD1) emerged as a highly upregulated gene in response to treatment with TP-3654. (D) Validation of the induction of DDIT4 by TP-3654.786-O, A498, Caki-1, Caki-2, Achn, and RCC4 cells were treated with the indicated concentrations of TP-3654 for 24 h. Gene expression levels were quantified using qRT-PCR and normalized to GAPDH. Mean ± SD, n = 3, * indicates significant difference from Control, p < 0.05. (E) Pharmacodynamic effects of TP-3654 on the AMPK/mTOR signaling cascade. 786-O and A498 cells were treated with the indicated concentrations of TP-3654 for 24 h. Protein lysates were subjected to immunoblotting to assess the effects of drug treatment on the expression of the following factors: phospho-AMPK (Thr172), total AMPK, REDD1, TXNIP, phospho-mTOR (Ser2448), total mTOR, phospho-p70S6K (Thr389), total p70S6K, phospho- 4E-BP1 (Thr37/46), total 4E-BP1, p62/SQSTM1, and LC3B. Tubulin documented equal protein loading.

Article Snippet: 786-O cells were infected with lentiviral particles containing GFP and a puromycin resistance gene (cat. # PS100093V) or a DDIT4 cDNA construct (cat. # RC202847L4V) according to the manufacturer’s protocol (Origene, Rockville, MD).

Techniques: Expressing, MTT Assay, Biomarker Discovery, Gene Expression, Quantitative RT-PCR, Control, Western Blot

Journal: Cancer letters

Article Title: REDD1 is a determinant of the sensitivity of renal cell carcinoma cells to autophagy inhibition that can be therapeutically exploited by targeting PIM activity.

doi: 10.1016/j.canlet.2025.217496

Figure Lengend Snippet: Fig. 4. DDIT4/REDD1 status determines sensitivity to autophagy inhibition. (A) DDIT4 was knocked down in 786-O RCC cells using lentiviral shRNA and knocked out in HAP1 cells using CRISPR. Knockdown/knockout efficiency was assessed for both cell lines by immunoblotting. (B) DDIT4 knockdown significantly reduces CQ- induced LC3B punctae formation. 786-O cells infected with control or DDIT4-targeted shRNA were treated with 25 μM CQ for 24 h LC3B punctae were visualized and quantified by immunocytochemistry. Mean ± SD, n = 10. * indicates a significant difference from shRNA controls, p < 0.05. (C) Genetic impairment of DDIT4 reduces cellular sensitivity to CQ. 786-O cells infected with control or DDIT4-targeted shRNA (left) and isogenic HAP1 cells with and without DDIT4 knockout (right) were treated with the indicated concentrations of CQ for 72 h. The impact of drug treatment on cell viability was determined by MTT assay. Mean ± SD, n = 3. * indicates a significant difference from shRNA controls or DDIT4 +/+ cells, p < 0.05. (D) Targeting DDIT4 blunts the pro-apoptotic effects of CQ. 786-O cells infected with control or DDIT4-targeted shRNA (left) and isogenic HAP1 cells with and without DDIT4 knockout (right) were treated with the indicated concentrations of CQ for 48 h. The impact of drug treatment on apoptosis induction was determined by staining with a FITC-tagged active caspase-3 antibody followed by flow cytometry. Mean ± SD, n = 3. * indicates a significant difference from shRNA controls or DDIT4 +/+ cells, p < 0.05. (E) REDD1 (DDIT4) overexpression synergistically enhances the sensitivity of RCC cells to autophagy inhibition. REDD1 was overexpressed in 786-O cells using a GFP-tagged lentiviral construct. Overexpression efficiency was assessed by immunoblotting (left). GFP and REDD1-GFP overexpressing cells were treated with the indicated concentrations of CQ for 72 h (middle, MTT) or 48 h (right, active caspse-3). The effects of drug treatment on cell viability (middle) and apoptosis (right) were quantified for each experimental condition. Mean ± SD, n = 3. * indicates a significant difference from GFP control cells, p < 0.05.

Article Snippet: 786-O cells were infected with lentiviral particles containing GFP and a puromycin resistance gene (cat. # PS100093V) or a DDIT4 cDNA construct (cat. # RC202847L4V) according to the manufacturer’s protocol (Origene, Rockville, MD).

Techniques: Inhibition, shRNA, CRISPR, Knockdown, Knock-Out, Western Blot, Infection, Control, Immunocytochemistry, MTT Assay, Staining, Flow Cytometry, Over Expression, Construct