human htert immortalized non transformed diploid retinal pigment epithelial rpe1 cells  (ATCC)

Journal: iScience

Article Title: Growth factor-independent mTORC1 signaling promotes primary cilia length via suppression of autophagy

doi: 10.1016/j.isci.2025.114204

Figure Lengend Snippet: mTORC1 inhibition shortens primary cilia (A–C) Cell number (A), flow cytometric analysis of cell cycle distribution via genomic incorporation of EdU as a function of 4',6-diamidino-2-phenylindole (DAPI)-based nuclear intercalation (B), and distribution of cell volumes in RPE1 cells treated with DMSO, fetal bovine serum (FBS) (10%), rapamycin, or Torin1 for the indicated times (C). (D) Confocal microscopy images of the immunofluorescent axoneme (acetylated tubulin in magenta) and basal body (γ-tubulin in cyan) of primary cilia in RPE1 cells treated with DMSO (left) or rapamycin (right) for the final 24 h. Regions of interest (dotted in top images) are shown at higher magnification in the bottom images as merged images (left) and single-channel grayscale for acetylated tubulin (right). Nuclei are stained with DAPI. Scale bars, 5 μm. (E and F) Primary cilia length (E) and percentage of ciliated cells in RPE1 cells treated with DMSO or rapamycin (F). (G) Time-course analysis of primary cilia length in RPE1 cells treated with rapamycin after 24 h of serum starvation. (H) Primary cilia length in RPE1 cells treated with rapamycin for varying durations within a total of 48 h of serum starvation. (I and J) Immunoblot (I) and primary cilia length (J) of RPE1 cells treated with DMSO, rapamycin, Torin1, leucine deprivation, or complete amino acid deprivation for the final 24 h in serum-free conditions (48 h total). (K) Schematic diagram of the generation of the GFP-Arl13B RPE1 cell line and live-cell imaging for primary cilia length measurement. (L) Primary cilia length of GFP-Arl13B RPE1 cells before Torin1 treatment, after 24 h of treatment with Torin1 (250 nM), and 24 h after Torin1 removal. All treatments were applied for the final 24 h in serum-free conditions (48 h total), unless otherwise specified: DMSO (0.1%), rapamycin (20 nM), and Torin1 (250 nM). Data are represented as mean ± standard deviation (SD). Statistical analysis was performed using two-way ANOVA followed by Šídák’s multiple comparisons test. The significance threshold (α) was set at 0.05, and all p values are reported.

Article Snippet: The hTERT-immortalized human retinal pigment epithelial (RPE1) cells (ATCC; CRL-4000) were cultured in RPMI-1640 supplemented with 10% FBS and 1% penicillin-streptomycin in a humidified incubator at 37°C with 5% CO 2 .

Techniques: Inhibition, Confocal Microscopy, Staining, Western Blot, Live Cell Imaging, Standard Deviation

Journal: iScience

Article Title: Growth factor-independent mTORC1 signaling promotes primary cilia length via suppression of autophagy

doi: 10.1016/j.isci.2025.114204

Figure Lengend Snippet: Genetic perturbation of mTORC1 signaling demonstrates that it promotes the elongation of primary cilia (A and B) Immunoblot (A) and primary cilia length (B) in doxycycline-inducible shScrambled and shMTOR RPE1 cells treated with doxycycline (0.5 μg/mL) for 7 days. (C) Immunoblot of isogenic clonal RPE1 cell lines with sgRNAs targeting TSC1, TSC2, or TBC1D7. Cells were serum-starved overnight. TSC1 and TSC2 are indicated by black arrowheads. (D) Primary cilia length in RPE1 lines used in (C). (E) Immunoblot of parental, sgTSC2, and human TSC2-rescued sgTSC2 RPE1 cells treated with Torin1 for 1 h. (F) Primary cilia length in RPE1 cell lines used in (E) following Torin1 treatment. (G) Immunoblot of Tsc2 +/+ , Tsc2 −/− , and human TSC2-rescued Tsc2 −/− MEFs after overnight serum starvation. (H) Primary cilia length in MEF cell lines used in (G). (I) Immunoblot of parental and sgNPRL2 RPE1 cells treated with overnight amino acid starvation followed by 1 h of amino acid stimulation. (J) Primary cilia length in parental and sgNPRL2 RPE1 cells treated with DMSO, Torin1, leucine deprivation, or complete amino acid deprivation. All treatments for primary cilia assessment were applied during the final 24 h in serum-free conditions (48 h total): DMSO (0.1%), rapamycin (20 nM), and Torin1 (250 nM). Data are represented as mean ± standard deviation (SD). Statistical analysis was performed using two-way ANOVA followed by Šídák’s multiple comparisons test. The significance threshold (α) was set at 0.05, and all p values are reported.

Article Snippet: The hTERT-immortalized human retinal pigment epithelial (RPE1) cells (ATCC; CRL-4000) were cultured in RPMI-1640 supplemented with 10% FBS and 1% penicillin-streptomycin in a humidified incubator at 37°C with 5% CO 2 .

Techniques: Western Blot, Standard Deviation

Journal: iScience

Article Title: Growth factor-independent mTORC1 signaling promotes primary cilia length via suppression of autophagy

doi: 10.1016/j.isci.2025.114204

Figure Lengend Snippet: mTORC1 signaling promotes elongation of primary cilia via autophagy suppression (A) Schematic diagram showing the generation of the GFP-LC3B-RFP autophagy reporter RPE1 cell line and flow cytometry analysis used to assess autophagic flux. (B and C) Flow cytometric analysis (B) and quantification of autophagic flux (C) in RPE1 cells treated with FBS (10%), bafilomycin A1, rapamycin, Torin1, or leucine deprivation for the indicated times. (D and E) Immunoblot (D) and primary cilia length (E) in RPE1 cells treated with DMSO, rapamycin, chloroquine, or bafilomycin A1. (F and G) Immunoblot (F) and primary cilia length (G) in RPE1 cells treated with DMSO, chloroquine, or bafilomycin A1, alone or in combination with rapamycin. (H) Primary cilia length in RPE1 cells treated with DMSO, ULK1/2 inhibitor (SBP-7455), or vacuoar protein sorting 34, class III PI 3-kinase (VPS34) inhibitor (VPS34-IN1). (I) Immunoblot of parental and sgATG7 RPE1 cells treated with bafilomycin A1 for the final 3 h under overnight serum starvation. p62 is indicated by a black arrowhead. (J) Primary cilia length in RPE1 cells used in (I) following treatment with DMSO, Torin1, or bafilomycin A1. (K) Immunoblot of parental and sgTFEB RPE1 cells (left) and primary cilia length in parental or sgTFEB RPE1 cells treated with DMSO, Torin1, or bafilomycin A1 (right). (L) Immunoblot of parental, sgTSC2, and human TSC2-rescued sgTSC2 RPE1 cells treated with bafilomycin A1 for the final 3 h under overnight serum starvation. (M) Primary cilia length in RPE1 cells used in (L) following treatment with DMSO or bafilomycin A1. All treatments were applied during the final 24 h in serum-free conditions (48 h total), unless otherwise specified: DMSO (0.1%), rapamycin (20 nM), Torin1 (250 nM), chloroquine (50 μM), bafilomycin A1 (100 nM), SBP-7455 (10 μM), and VPS34-IN1 (10 μM). Data are represented as mean ± standard deviation (SD). Statistical analysis was performed using two-way ANOVA followed by Šídák’s multiple comparisons test. The significance threshold (α) was set at 0.05, and all p values are reported.

Article Snippet: The hTERT-immortalized human retinal pigment epithelial (RPE1) cells (ATCC; CRL-4000) were cultured in RPMI-1640 supplemented with 10% FBS and 1% penicillin-streptomycin in a humidified incubator at 37°C with 5% CO 2 .

Techniques: Flow Cytometry, Western Blot, Standard Deviation