Fig 2: Schematic model summarizing our findings regarding the contribution of PACS-2 in ER-phagy and HG-induced renal tubular injury.PACS-2 is a membrane trafficking protein that promotes TFEB nuclear translocation, and thus inducing Fam134b gene transcription and facilitating ER-phagy process by interacting with LC3β. Under diabetic conditions, down-regulated PACS-2 decreases TFEB translocation and hence affects Fam134b gene transcription, followed by the decrease of the interaction between FAM134B and LC3β. ER-phagy is then blocked, leading to the accumulation of damaged or excessive ER in cytoplasm, and ultimately contributing to the activation of inflammatory response and the occurrence of fibrosis. LIR LC3-interacting region.

Fig 3: FAM134B flux can be monitored using the SRAI probe. (A) Schematic representing some of the characterized ER-phagy cargo receptors. LIR, LC3 interacting region; AIM, ATG8 interaction region; RHD, reticulon homology domain; TMD, transmembrane domain. (B) Schematic of the expected fluorescence profile of SRAI (TOLLES-YPet)-FAM134B within different compartments (representing TOLLES as a FRET donor and YPet as a FRET acceptor). TOLLES is represented in blue and YPet in yellow or faint yellow if quenched. (C) Imaging-based autophagic flux assay in PDAC cells stably expressing SRAI-FAM134B (transfected with sgControl or sgAtg7) in full nutrients (untreated) or following 4 h starvation in the presence or absence of BafA1 (50 nM) and imaged using automated widefield microscopy. Representative immunofluorescence images of TOLLES (magenta) and YPet (green). Bar = 50 µm. On the bottom right, quantification of the TOLLES:YPet index (n = 3, 6 fields per replicate were analyzed with 60–100 cells per field, ±S.D., * = p < 0.05, ** = p < 0.01, 1-way ANOVA followed by Holm-Šídák comparisons). (D) Immunofluorescence images of PDAC cells treated in the presence or absence of BafA1 (50 nM, 4 h). Cells were fixed and stained for FAM134B and imaged by confocal microscopy. Bar = 50 µm.

Fig 4: Role of SFKs in the regulation of ER homeostasis. (A) SFK overexpression suffices to induce ER expansion in an enzymatic activity-related fashion. Lck- or Lyn-expressing cells and the -Dox counterparts were stained with ER tracker Blue-White DPX at the indicated time points after Dox addition to the cell cultures and analyzed by FACS. ER expansion measured by increases of ER tracker dye fluorescence (lower panel histograms) is correlated to increases in SFK protein levels (GFP fluorescence, upper panel histograms). 18h incubation with 30μM of the pan SFK inhibitor PP2 reduced ER expansion to baseline (-Dox) levels. The correspondent reduction of pY416 after PP2 incubation for each SFK is shown in the bottom-panel histograms. Treatment with 50 µM of the ER-stress inducer Chloroquine (CQ) for 12h was used a positive control for the expected function of the ER-tracker reagent. Graph shows the ratio of ER tracker dye MFI from Lck- or Lyn-expressing cells to -Dox treated cells. Since essentially there was no difference between the Lck and Lyn samples, statistics were calculated for the mean values between each time point (n=4, Unpaired Student t test; mean +/- SD; **P < 0.01, ***P < 0.001; ns, not significant). (B) SFK-driven ER expansion is not concurrent with upregulation of UPR mediators. RT-qPCR of the indicated UPR modulators. Each time point corresponds to samples stained with ER tracker shown in (A) RT-qPCR data are shown log2FC of the gene expression between Lck-BJAB (black)/-Dox and Lyn-BJAB (grey)/-Dox (n=2, Multiple unpaired Student t test; mean +/- SD; *P < 0.05, **P < 0.01, ***P < 0.001; ns: not significant). (C) SFK overexpression suffices to induce FAM134B oligomerization in an enzymatic activity-related fashion. Lck- or Lyn-expressing cells and their -Dox counterparts were mixed at a ratio of 1:1, immobilized on poly-D-lysine coated microscope slides, stained for FAM134B (white), and visualized by confocal microscopy. GFP (green) denotes the SFK-expressing cells. Sample sets include: i) untreated cells (left-hand side panels) ii) SFK-expressing cells treated with 30μM PP2 for 18h, mixed with untreated -Dox cells (right-hand side panels) to provide a comparative visualization of SFK activity influence on the cargo receptor clustering and iii) -Dox cells treated with 50 µM CQ for 12h (indicative image shown in lower panels of D, below). FAM134B oligomers within single cells, appearing as distinctive puncta, were quantified for each sample. Collective quantitation results from three independent experiments (for the untreated samples) and two independent experiments (for PP2 and CQ treatments) are displayed on the adjacent graph. Number of cells for each sample -Dox/untreated n=239, -Dox/CQ n=79, Lck-BJAB/untreated n=164, Lck-BJAB/PP2 n=98, Lyn-BJAB/untreated n=144, Lyn-BJAB/PP2 n=84. Scale bar, 5 µm (n=2, Unpaired Student t test; mean +/- standard deviation [SD]; ****P < 0.0001). (D) SFK-driven FAM134B oligomerization does not coincide with LC3 colocalization. Lck- or Lyn-expressing cells and their -Dox counterparts mixed at a ratio of 1:1 (upper panels), or -Dox cells treated with 50 µM CQ for 12h (lower panels) were immobilized on poly-D-lysine coated microscope slides, stained for FAM134B (white) and LC3B (red), and visualized by confocal microscopy. GFP (green) denotes the SFK-expressing cells. Colocalization of FAM134B and LC3 was quantified by the coloc2 pre-installed plugin of Image (J) Collective colocalization analysis results from two independent experiments are displayed on the adjacent graph. Number of cells for each sample -Dox/untreated n=116, -Dox/CQ n=116, Lck-BJAB/untreated n=76, Lyn-BJAB/untreated n=73. Scale bar, 5 µm (n=2, Unpaired Student t test; mean +/- standard deviation [SD]; ****P < 0.0001).

Fig 5: Exacerbated ERphagy induced by the combination of NU7441 and CV-B5/F degraded STING activated by nuclear cGAS recruited by DSBs. a NCI-H460 was treated with 1 μM NU7441, 0.01 MOI CV-B5/F, or a combination for 24 h. Immunoblots of anti-viral proteins including IRF9 and p-STAT1 were detected. b NCI-H460 cells were treated with 1 μM NU7441, 0.01 MOI CV-B5/F, or a combination for 24 h. FAM134B (a marker of ERphagy) and STING were determined by western blot. c NCI-H460 cells knocked down with shRNA for RIG-I were treated with 1 μM NU7441, 0.01 MOI CV-B5/F, or a combination for 24 h. RIG-I, FAM134B and STING were determined by western blot. Gray values of NC, shRNA-1 and shRNA-2 were compared separately. d NCI-H460 cells overexpressing RIG-I were treated with 1 μM NU7441, 0.01 MOI CV-B5/F, or a combination for 24 h. RIG-I, FAM134B and STING were determined by western blot. Gray values of NC, OE-1 and OE-2 were compared separately. e, f NCI-H460 cells were treated with 1 μM NU7441 or 1 μM KU60019, 0.01 MOI CV-B5/F, or a combination for 24 h. Immunofluorescence of cGAS of nuclear was analyzed by confocal microscopy. Mean gray value of cGAS in nuclear and cytoplasma was separately analyzed by Image J software (n = 20)