Dm6000b

Droplet formation of protein samples was monitored by DIC microscopy. 15 μl of sample were loaded onto glass coverslips and DIC images were acquired on a DM6000B (Leica) microscope with a 63×-objective (water immersion). Alexa-488-labeled protein was prepared using Alexa Fluor 488 microscale protein labeling kit (Thermo Fisher Scientific). To confirm the presence of protein in the droplets, Alexa-488-labeled protein was mixed with unlabeled protein and the images were acquired on a DM6000B (Leica) microscope with a 63×-objective (water immersion) using a 488 nm argon laser line. Images were processed using ImageJ.

In this study, EdU was detected in all immunofluorescence experiments to allow identification of S phase cells. Cells were treated with 10 μM EdU for 30 min before fixation to label the S phase cells. At each time point after IR, the EdU-treated cells were washed once with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde in PBS for 10 min at 4 °C, followed by permeabilization with 0.5% Triton X-100 in PBS for 5 min at 4 °C. Primary antibodies were then applied for 30 min at 37 °C, followed by reaction with Alexa 488- or 555-conjugated secondary antibodies (Life Technologies, USA) for 30 min at 37 °C. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). All of the antibodies were diluted in 5% skim milk/Tris-buffered saline with 0.1% Tween 20.
After immunofluorescence staining, EdU was detected following the manufacturer’s protocol. Images were acquired using a fluorescence microscope (DM6000B, Leica, Germany) or a confocal fluorescence microscope (LSM800, Zeiss, Germany). For image acquisition using DM6000B (Leica), x63 or x100 objective lenses, CCD camera (Orca R2, Hamamatsu Photonics), and FW4000 software were used. For image acquisition using LSM800 (Zeiss), x63 or x100 objective lenses, GaAsP detector, and ZEN 2.1 image acquisition software were used.

The removed kidneys were fixed with 4% paraformaldehyde, embedded in paraffin and then cut into 4-μm-thick sections. Periodic acid–Schiff (PAS, Sigma-Aldrich, USA) staining was performed on the sections for histologic analysis. Glomerulosclerosis scoring was assessed blindly on a minimum of 10 glomeruli per section using a light microscopy (Leica DM 6000B; Leica Microsystems, Wetzeler, Germany).
Immunohistochemical staining for METTL14 (HPA038002, Sigma), WT1 (sc-7385, Santa Cruz Biotechnology) and Sirt1(ab110304, Abcam) was performed. Briefly, after dewaxing, subjected to antigen retrieval and blocked, the sections were incubated with the primary antibodies overnight at 4 °C. The next day, positive staining was revealed by incubating with HRP-conjugated secondary antibodies, diaminobenzidine staining and counterstaining with hematoxylin. Slides were visualized by a light microscopy (Leica DM 6000B; Leica Microsystems, Wetzeler, Germany).

Human breast cancer cells were seeded 2 ×  ${10}^5$ cells on 16 mm glass coverslips in 6 well plates with the culture medium for 24 h and then starved in serum-free DMEM-F12 for 16 h. hUCMSCs were seeded 1 ×  ${10}^5$ cells in 0.4 μm pore, 24 mm transwell inserts with the culture medium for 24 h and starved for 16 h in DMEM-LG medium with 1% FBS. Then breast cancer cells were pretreated with 50 μM embelin and hUCMSCs were pretreated with100 ng/ml IL-1β for 24 h. After pretreatment, the transwell inserts were moved into 6 well plates to co-culture breast cancer cells and hUCMSCs for 24 h in DMEM-LG with 1% FBS. The coverslips were removed and Annexin V-FITC Apoptosis Detection Kit (Strong Biotech Corporation, TPE, ROC) were used to detect apoptosis. Breast cancer cells were stained with 2% Annexin V-FITC (AV) and 2% Propidium Iodide (PI) in a binding buffer for 10 min in the dark. After staining, the images were immediately captured by using a Fluorescent Microscope (Leica DM6000B, Wentzler, Germany). The apoptotic rate in Fig. 5 was determined by calculating the proportion of cells that have undergone apoptosis (cell numbers of Annexin V+/PI−, Annexin V+/PI+ expressing cells) in relation to the total number of cells in five randomly selected fields of three independent experiments.