Ab10646

Immunohistochemistry staining was performed using standard techniques, as described previously.17 Sections were incubated with VEGF‐A (ab1316; Abcam), FGF‐2 (ab181; Abcam), FGF receptor (FGFR)‐1 (ab10646; Abcam), FGFR‐2 (ab10648; Abcam), PDGF‐BB (ab178409; Abcam), PDGFR‐β (3169; Cell Signaling Tchonology), CD31 (ab9498, ab222783; Abcam), neuron‐glial antigen 2 (NG2, ab129051; Abcam,), anti‐glycophorin A (ab194397; Abcam,) and α‐SMC actin (ab7817, Abcam; 19245, Cell Signaling Tchonology) antibody at 4°C. Alexa Fluor 594 secondary antibody (anti‐rabbit IgG, 8889S), Alexa Fluor 555 secondary antibody (antimouse IgG, 4409S), Alexa Fluor 488 secondary antibody (antimouse IgG, 4408S; anti‐rabbit IgG, 4412S) and DAPI were used. Images were visualized by laser scanning confocal microscopy (LSM710; Zeiss).

Deparaffinized sections or fixed cells were permeabilized, blocked and incubated with acetylated α-tubulin (1:200, T6793, Sigma) overnight at 4. The slides were washed and stained with Alexa Fluor 568 conjugated anti-rabbit IgG (1:1000, Invitrogen) antibody for 1 hour at room temperature. DAPI (6-diamidino-2-phenylindole, Sigma) staining was used as the counterstain for nuclei.
The same staining procedure was used for acetylated α-tubulin (1:500, T6793, Sigma), FGFR1 (1:100, ab10646, Abcam), p-FGFR1 (1:100, ab59194, Abcam), Phospho-SMAD1 (Ser206) (1:100, PA517092, Invitrogen), and p-Smad1/5/8 (1:50, sc-12353-R, Santa Cruz) staining.

Primary antibodies anti-FGF21 (ab171941, 1:300 dilution), anti-FGFR1 (ab10646, 1:200 dilution), anti-leptin (ab3583, 1:200 dilution), and anti-HSL (ab220074, 1:200 dilution) were obtained from Abcam (UK). Secondary antibodies, including SABC anti-rabbit and anti-mouse IgG (1:500 dilution), were purchased from Bioss (Beijing, China).

Chromosome Conformation Capture (3C) samples were prepared following methods previously described in [5 (link),71 (link)] with minor changes using Hind III for restriction digestion (10798983001 Roche, Branchburg, NJ, USA). Chromatin Immunoprecipitation (ChIP) samples were collected and prepared using the MAGnify Chromatin Immunoprecipitation System Kit (492024 ThermoFisher, Waltham, MA, USA) with minor alterations. Immunopreciptations were completed using FGFR1 ab10646 Abcam, Cambridge, MA, CTCF ab70303 Abcam, Cambridge, MA, USA and control Rb IgG MAGnify 492024 ThermoFisher, Waltham, MA, USA. Samples for mRNA were collected using the RNeasy Mini Kit (74104 Qiagen, Germantown, MD, USA) with minor alterations. Primer designs are shown in Table S1 Sheet 3. Three technical replicates were completed on the same plate for each biological replicate measurement. Three biological replicates were completed for 3C-qPCR, five for FGFR1 ChIP-qPCR, three for CTCF ChIP-qPCR, and three for RT-qPCR.
3C-qPCRs measurements were calculated by Percent Input of a non-digested location
(at the GapDH gene):
Percent Input = [100 * 2⌃(GapDH CT − Interaction CT)] * 100
ChIP-qPCR measurements were calculated as Percent 10% Input:
Percent Input = 100 * 2⌃(10% Input CT − IP sample CT)
RT-qPCR measurement were calculated as Relative Expression:
Relative Expression = [100 * 2⌃(−1 * cDNA CT)] * 10⁶

Animal treatments were approved by the Animal Ethics Committee of Hainan general hospital(Hainan affiliated hospital of Hainan medical university). Ten male BALB/C nude mice (5–6 weeks old, 12 ± 4 g) were obtained from Hunan SJA Laboratory Animal Co., Ltd. (Changsha, China). Stably knocked down A549 cells and control cells (approximately 1 × 10⁷) were injected subcutaneously into the axilla of BALB/C nude mice. Width and length were measured with calipers from day 3 after transplantation, and tumor growth was monitored weekly, and tumor volume was calculated: (Width² × length)/2. After 4 weeks, mice were euthanized and tumor weights were measured. Tumors were assayed for Ki-67 and FGFR1 expression using immunohistochemistry as previously described using antibodies Ki-67 (ab16667, 1:100, Abcam) and FGFR1 (ab10646, 1:1500, Abcam) [29 (link)].

Total protein extracts were obtained by 500 μl RIPA lysis buffer (Beyotime, Shanghai, China). Protein (20 μg) was loaded onto 8% sodium dodecyl sulphate polyacrylamide gel electrophoresis (Solarbio, Beijing, China). Afterward, the sample was transferred to polyvinylidene fluoride membranes (Invitrogen) and blocked with 5% skim milk. Primary antibodies rabbit FGFR1 (ab10646, 1:10,000, Abcam), Bax (1:1000, 50,599–2-IG, Proteintech), Bcl-2 (1:1000, sc-7382, Santa Cruz Biotechnology), and GAPDH (ab9485, 1:2500, Abcam) were incubated overnight at 4℃, and horseradish peroxidase-conjugated goat anti-rabbit secondary antibody IgG (1:1000, ab181236, Abcam) was then detected for 2 h. Signals were developed by ECL kit (34,080, Thermo Fisher Scientific) and analysis was done by ImageJ software [22 (link)].